Rad51-like: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Rad51-like/spindle A
Synonyms - DMRad51. Rad51-like
Cytological map position - 99-D3
Function - enzyme
Symbol - Rad51/spn-A
FlyBase ID: FBgn0011700
Genetic map position -
Classification - DNA dependent ATPase activity
Cellular location -
|Recent literature||Alexander, J. L., Beagan, K., Orr-Weaver, T. L. and McVey, M. (2016). Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27849606
Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. Drosophila follicle cell developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. This system was used to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. A null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase theta/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, these findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context.
|Ryu, T., Bonner, M. and Chiolo, I. (2016). Cervantes and Quijote protect heterochromatin from aberrant recombination and lead the way to the nuclear periphery. Nucleus [Epub ahead of print] PubMed ID: 27673416
Repairing double-strand breaks (DSBs) is particularly challenging in heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination and chromosome rearrangements. In Drosophila cells, faithful homologous recombination (HR) repair of heterochromatic DSBs relies on a specialized pathway that relocalizes repair sites to the nuclear periphery before Rad51 recruitment. This study shows that HR progression is initially blocked inside the heterochromatin domain by SUMOylation and the coordinated activity of two distinct Nse2 SUMO E3 ligases: Quijote (Qjt) and Cervantes (Cerv). In addition, the SUMO-targeted ubiquitin ligase (STUbL) Dgrn, but not its partner dRad60, is recruited to heterochromatic DSBs at early stages of repair and mediates relocalization. However, Dgrn is not required to prevent Rad51 recruitment inside the heterochromatin domain, suggesting that the block to HR progression inside the domain and relocalization to the nuclear periphery are genetically separable pathways. Further, SUMOylation defects affect relocalization without blocking heterochromatin expansion, revealing that expansion is not required for relocalization. Finally, nuclear pores and inner nuclear membrane proteins (INMPs) anchor STUbL/RENi components and repair sites to the nuclear periphery, where repair continues. Together, these studies reveal a critical role of SUMOylation and nuclear architecture in the spatial and temporal regulation of heterochromatin repair and the protection of genome integrity.
|Khan, C., Muliyil, S., Ayyub, C. and Rao, B. J. (2020). spn-A/rad51 mutant exhibits enhanced genomic damage, cell death and low temperature sensitivity in somatic tissues. Chromosoma. PubMed ID: 33222024
Homologous recombination (HR) is one of the key pathways to repair double-strand breaks (DSBs). Rad51 serves an important function of catalysing strand exchange between two homologous sequences in the HR pathway. In higher organisms, rad51 function is indispensable with its absence leading to early embryonic lethality, thus precluding any mechanistic probing of the system. In contrast, the absence of Drosophila rad51 (spn-A/rad51) has been associated with defects in the germline, without any reported detrimental consequences to Drosophila somatic tissues. A systematic analysis was performed of developmental defects in somatic tissues of spn-A mutant flies by using genetic complementation between multiple spn-A alleles. This study uncovers a requirement for spn-A in somatic tissue maintenance during both larval and pupal stages. Also, this study shows that spn-A mutant exhibits patterning defects in abdominal cuticle in the stripes and bristles, while there appear to be only subtle defects in the adult wing and eye. Interestingly, spn-A mutant shows a discernible phenotype of low temperature sensitivity, suggesting a role of spn-A in temperature sensitive cellular processes. In summary, this study describes the important role played by spn-A/rad51 in Drosophila somatic tissues.
Five spindle genes, spn-A, spn-B, spn-C, spn-D and spn-E were originally identified in a screen for maternal-effect mutants on the third chromosome because homozygous mutant females lay ventralized eggs (Tearle, 1987). spn-A has been identified as the Drosophila Rad51-like (Rad51) gene, whose sequence among the five known Drosophila Rad51-like genes is most closely related to the Rad51 homologs of human and yeast. Rad51 is a conserved protein essential for recombinational repair of double-stranded DNA breaks (DSBs) in somatic cells and during meiosis in germ cells. Yeast Rad51 mutants are viable but show meiosis defects. In the mouse, RAD51 deletions cause early embryonic death, suggesting that in higher eukaryotes Rad51 is required for viability. Drosophila Rad51/spn-A null mutants are viable but oogenesis is disrupted by the activation of a meiotic recombination checkpoint. The meiotic phenotypes result from an inability to effectively repair DSBs. In Drosophila the Rad51-dependent homologous recombination pathway is not essential for DNA repair in the soma, unless exposed to DNA damaging agents. It is therefore proposed that under normal conditions a second, Rad51-independent, repair pathway prevents the lethal effects of DNA damage (Staeva-Vieira, 2003).
Chromosomal integrity is essential for proper embryonic and postembryonic development, prolonged survival and successful reproduction. Highly conserved repair mechanisms exist in all organisms, from bacteria to mammals, to recognize and repair DNA damage. The repair of double-stranded DNA breaks (DSBs) is a necessary mechanism for recombining parental genomes during meiosis and is used as a defense mechanism after DNA damage caused by irradiation or chemical agents. The presence of DNA damage activates a cell cycle checkpoint. This allows time for the cell to correct the damage so as not to propagate the defect or affect normal cellular functions. In Saccharomyces cerevisiae mutations in the same genes show increased sensitivity to ionizing radiation and meiotic phenotypes, such as chromosome non-disjunction and/or rearrangements. This suggested a functional relationship between the mechanisms of mitotic DNA repair and meiotic recombination (Staeva-Vieira, 2003).
Genetic studies in S.cerevisiae led to the discovery of the Rad52 epistasis group of DSB repair genes. A core protein in this pathway is Rad51, which is related to the bacterial RecA protein. Rad51 has DNA-dependent ATPase activity and catalyzes strand exchange between homologous DNA molecules. Rad51 and Rad51-related proteins are found from yeast to humans (for review see Sung, 2003). In yeast, the Rad51 null mutant is viable but shows sporulation defects (Shinohara, 1992). The mouse Rad51 knockout is embryonic lethal (Lim, 1996; Tsuzuki, 1996), thus the role of Rad51 in mouse meiosis could not be studied. In both mouse and yeast, a meiosis-specific Rad51-related gene, Dmc1 (Bishop, 1992; Pittman, 1998), has been identified and shown to be required for chromosome synapsis and strand exchange during prophase of meiosis I (Staeva-Vieira, 2003).
In contrast to yeast, Drosophila members of the Rad52 epistasis group were not identified on the basis of meiosis defects or mutagen sensitivity. Rather, mutations in the Drosophila RAD51-related gene, spindle-B (spnB), and the Rad54 homolog, okra, were discovered as maternal-effect mutants with altered patterning of the eggshell, the so-called spindle phenotype (Morris, 1999). It was shown that this phenotype, observed in spnB and okra mutants, was due to reduction in the levels of the morphogen Gurken, a TGFalpha-like protein that controls both dorso-ventral patterning of the egg and antero-posterior polarity of the embryo (Ghabrial, 1998). Ghabrial suggested that the activation of a meiotic checkpoint, which resulted in defective Gurken translation, was the result of a failure to repair DNA breaks in mutants for okra, spnB and spindle-D (spnD), another Rad51-related protein (Ghabrial, 1999; Abdu, 2003). Accordingly, the spindle phenotype was suppressed by mutants for the Spo11 homolog, mei-W68 -- these mutants are defective in double-stranded break formation and thus are unable to activate the checkpoint (Ghabrial, 1999). Spn mutants were also suppressed in combination with mutants of known transducers of cell cycle checkpoints, such as Drosophila mei-41, an ATR/ATM phosphatidylinositol 3-kinase-like protein, and the Drosophila homolog of Chk2 kinase, chk2/mnk/loki (Ghabrial, 1999; Abdu, 2002). A target for the meiotic checkpoint in Drosophila is the ATP-dependent helicase Vasa (Styhler, 1998; Tomancak, 1998), which is phosphorylated upon checkpoint activation and may regulate Gurken translation (Ghabrial, 1999). Sequence analysis indicates that there are at least five Drosophila genes that show significant homology to yeast and human Rad51. It remained unclear whether these genes have distinct functions in DSB repair and whether the activation of the meiotic checkpoint was a consequence of the failure to repair DSBs. Furthermore, while mutations in spnB show meiotic defects, they do not affect DNA repair in somatic cells (Ghabrial, 1998), raising the possibility that in Drosophila distinct sets of Rad51-like genes may control DSB repair either in the germline or in the soma (Staeva-Vieira, 2003).
spnA mutants exhibit the spindle eggshell phenotype. In spnA oocytes synapse of homologous chromosomes is correctly initiated during meiosis but its resolution is delayed and unrepaired double-stranded breaks persist longer than in wild type causing the activation of a meiotic recombination checkpoint. spnA null mutants are viable but show sensitivity to irradiation, suggesting that SpnA acts in the soma but that other repair mechanisms compensate in the absence of SpnA. Analysis of the expression pattern of the five known Drosophila Rad51 homologs together with the analysis of the mutant phenotype of three of these genes suggest that the Drosophila Rad51 genes act in concert during oogenesis and that only a subset of them are used for repair in the soma (Staeva-Vieira, 2003).
Screens in Drosophila have recovered many mutations that cause disruption to normal meiotic chromosome behavior. They were identified based on the ability to recognize abnormal events, such as chromosome loss, non-disjunction or a change in recombination frequency. Mutagen sensitivity screens, similar to those performed in yeast, have also been conducted in Drosophila to identify genes necessary for DNA repair. As would be expected, some of these mutagen-sensitive mutants showed meiotic defects as well. Interestingly, none of the Rad52 epistasis genes of Drosophila were recovered from these types of screens. Instead, due to downstream effects on D/V patterning through the activation of a meiotic checkpoint, the spindle oogenesis phenotype has proven to be an effective assay by which to uncover these genes. Thus far, four members of the Rad52 epistasis group in Drosophila have been found through this approach (Staeva-Vieira, 2003 and references therein).
In Drosophila, there are five members of the Rad51 family. This analysis confirms that Spindle-A is the structural and functional homolog of the yeast and mammalian Rad51 protein. Biochemical analysis of in vitro purified Rad51 has shown that it has strand exchange capabilities (Alexiadis, 2002). The other Rad51 paralogs show greater sequence homology to Rad51 accessory proteins, which have been shown to promote Rad51 foci formation on DNA. Both rad51D and spnD, in the adult, are expressed specifically in the germline. Therefore, it is suggested that they are Rad51 accessory proteins involved in meiotic recombination, compensating for a lack of a Drosophila Dmc1 homolog. Initial studies on spnB revealed a striking similarity to Dmc1, namely its importance in meiotic recombination and its resistance to the effects of MMS (Ghabrial, 1998). However, spnB RNA is expressed in the soma as well as the germline. Moreover, evidence is presented that spnB mutant larvae are less tolerant than their wild-type siblings to the DNA damaging effects of ionizing radiation. Based on its sequence homology to XRCC3, it is possible that SpnB functions as a necessary partner for Drosophila Rad51 during meiotic recombination and takes on a supporting role in Rad51 stabilization (Liu, 1998; Brenneman, 2002) during DSB repair of the soma (Staeva-Vieira, 2003).
Analysis of Rad51 function in vertebrate development has b een difficult due to the early embryonic lethality of RAD51-/- mice. Vertebrate cell culture studies have suggested an essential role of RAD51 in the repair of breaks generated during DNA replication (Sonoda, 1998), thus providing some explanation for the embryonic lethality in mice. Drosophila Rad51 null animals can survive to adulthood. Therefore, the requirement for Rad51 in the repair of DNA breaks occurring during DNA replication may not be conserved. However, other possibilities exist. (1) Maternal Rad51 may persist to repair DSBs occurring throughout embryogenesis. However, female flies doubly mutant for mei-W68 and spnA produce embryos that survive to adulthood, suggesting that neither maternal nor zygotic Drosophila Rad51 function are essential for viability. (2) Another possibility is that the Rad51 genes may have partially overlapping, redundant functions. However, neither of the other family members shows strong homology to Rad51. (3) Flies doubly mutant for the spnA and its closest relative, spnB, are viable (Gonzalez-Reyes, 1997) and the next closest paralog, spnD, is expressed specifically in the germline, though only adult animals have been tested. An alternative explanation, and the one that is favored, is the existence of an alternative repair pathway that can compensate in the event of homologous recombination failure. Homologous recombination has been considered the major DNA repair pathway in Drosophila. Recent evidence in Drosophila has shown that when the homologous recombination pathway is compromised, the error-prone non-homologous end joining (NHEJ) pathway can compensate and prevent a lethal outcome (Adams, 2003). Therefore, in Drosophila, it would be predicted that an efficient cooperation must exist between the homologous recombination and NHEJ pathways to prevent the lethal effects of DNA DSBs, presumably with homologous recombination being the primary choice and NHEJ playing a backup role (Staeva-Vieira, 2003).
During meiotic recombination, crossing over between homologous chromosomes guarantees their proper segregation. Defects in the proper formation of recombination intermediates result in the activation of a pachytene, or meiotic recombination, checkpoint. In mice, if defects in chromosomal synapsis or meiotic recombination persist, the result is the activation of the pachytene checkpoint and removal of the arrested germ cells most probably by apoptosis. In this study, it is shown that a meiotic recombination checkpoint is activated in response to a loss of SpnA function. spnA mutant females do not show an appreciable defect in egg deposition, suggesting that the apoptotic pathway is not activated in response to the meiotic recombination checkpoint. Moreover, the p53 protein, a strong inducer of apoptosis during the mitotic cell cycle, has been shown not to be involved in the Drosophila meiotic recombination checkpoint (Abdu, 2002). Instead, as the data indicate, the unsuccessful processing of meiotic-induced DSBs results in a Chk2-dependent delay of the meiotic cell cycle. Concomitant with this delay, a defect in the EGFR/TGFalpha signaling pathway is observed, that results in the production of eggs with dorsal/ventral patterning defects. Thus, these results show a coupling between progression through the meiotic cell cycle and oocyte patterning and development. The ATP-dependent helicase Vasa has been implicated in mediating at least two aspects of meiotic checkpoint activation, Gurken translation and karyosome formation. It remains unclear if Vasa is directly activated by the checkpoint transducer kinase Chk2/Mnk and how defects in DSB repair lead to checkpoint activation. The spindle eggshell phenotype has proven to be an efficient assay to identify genes that lead to the activation of the meiotic checkpoint, making Drosophila an excellent genetic system to identify additional components that regulate the interplay between DNA repair, cell cycle progression and cell differentiation during meiosis and possibly, as these studies suggest, also mitosis (Staeva-Vieira, 2003).
Telomeres protect chromosome ends from being repaired as double-strand breaks (DSBs). Just as DSB repair is suppressed at telomeres, de novo telomere addition is suppressed at the site of DSBs. To identify factors responsible for this suppression, an assay an assay was developed to monitor de novo telomere formation in Drosophila, an organism in which telomeres can be established on chromosome ends with essentially any sequence. Germline expression of the I-SceI endonuclease resulted in precise telomere formation at its cut site with high efficiency. Using this assay, the frequency of telomere formation was quantified in different genetic backgrounds with known or possible defects in DNA damage repair. It was shown that disruption of DSB repair factors (Rad51 or DNA ligase IV) or DSB sensing factors (ATRIP or MDC1) resulted in more efficient telomere formation. Interestingly, partial disruption of factors that normally regulate telomere protection (ATM or NBS) also led to higher frequencies of telomere formation, suggesting that these proteins have opposing roles in telomere maintenance vs. establishment. In the ku70 mutant background, telomere establishment was preceded by excessive degradation of DSB ends, which were stabilized upon telomere formation. Most strikingly, the removal of ATRIP caused a dramatic increase in telomeric retrotransposon attachment to broken ends. This study identifies several pathways that suppress telomere addition at DSBs, paving the way for future mechanistic studies (Beaucher, 2012).
In this study de novo telomere formation was induced upon an endonuclease induced DSB. Remarkably, as high as 63% of the progeny on average had acquired a new telomere at the DSB site under continuous I-SceI production through development starting from the earliest stages of embryonic divisions. This high rate of terminal deficiency (TD) recovery in the germline is in startling contrast to Muller's inability to recover terminally deleted chromosomes in Drosophila that had led to the very concept of “telomere” (Muller 1940, Muller and Herkowitz 1954) (Beaucher, 2012).
Four lines of explanation are offered for reconciliation. First, Muller used Xray irradiation as the DSB inducing agent whereas this study used a site-specific endonuclease. Breaks generated by irradiation might need to be processed differently or more extensively than ends from a nuclease digestion to become suitable substrates for telomere formation. Secondly, the DSB at telomeric marker D4A is relatively close to an existing telomere. It is possible that there is a higher concentration of capping proteins surrounding the telomeres in the nucleus making it more likely for a DSB end to be capped as a telomere. Thirdly, I-SceI induces one DSB per diploid genome in this system whereas the number of breaks induced by X-ray was difficult to control and some cells might have more than one. Cells respond differently to DSB dosage. Yeast cells in the G1 phase respond differently to one versus four DSBs induced by a nuclease. This different response might lead to inefficient telomere formation when a cell encounters more than one break. Although the above three factors might contribute to the decreased likelihood of recovering TDs in the Drosophila germline, they remain a priori assumptions to explain Muller's results since TDs can be nevertheless recovered in the female germline by X-ray irradiation (Beaucher, 2012).
A fourth proposition is offered as a key difference between the current experiments and those of Muller's: Muller induced DSBs to male germ cells in advanced stages of spermatogenesis yet the DSB induced in the current assays are limited to the mitotic compartment of the male germline. It was recently discovered that paternal telomeres that have lost the protection of the K81 capping protein engage in highly efficient telomere fusion before the first zygotic division (Gao, 2011). This result implies that first, de novo telomere establishment is highly inefficient on decondensed sperm DNA even in the presence of abundant maternal deposition of capping components; second, DNA repair, particularly end joining, is highly active during the early embryonic cycles. In Muller's experiments, the DSB generated in the male germline likely persist until after fertilization making it unlikely to be capped during the first zygotic division. On the contrary, the DSB in the current assay can be generated throughout development as I-SceI is continuously and ubiquitously expressed. In addition, simple rejoining of an I-SceI-induced DSB or inter-sister GC repair would recreate a functional cut site allowing a second round of cutting. Therefore, the DSB at D4A had multiple opportunities to acquire a telomere during all stages of development. Evidence supporting the last proposition already exists. In light of the increase in TD recovery from irradiated mu2 females, it has been postulated that DNA lesions induced in mu2 oocytes persist through oogenesis followed by telomere establishment on DSBs in the early embryo. A similar increase was not observed when sperm instead of oocytes were irradiated, suggesting DNA lesions on sperm chromatin are poor substrates for telomere formation. In addition, neo-telomere formation on ends of broken dicentric chromosomes can be readily recovered when induced in the mitotic male germline (Beaucher, 2012).
Evidence is also available suggesting that de novo telomere formation can occur very early in the somatic lineages. This was derived from scoring flies that inherited both D4A and a maternal I-SceI gene. In this assay, TD formation leads to the loss of the dominant KrIF mutation restoring the eye to its normal size. In addition, TD formation leads to a variegated eye pigmentation pattern. Flies were often recovered that had variegated eyes with sizes that are fully normal. These eyes likely developed from a cell with D4ATD formed at early stages of development (Beaucher, 2012).
Defective checkpoints allow telomere formation on persisting DSBs The first set of mutants that increase TD recovery in the germline have defects in DNA checkpoint functions: mu2 and mus304. From germlines in both backgrounds, increases were recorded of TD formation that were among the highest in all mutants tested. It is possible that cells able to establish telomeres on DSBs had a survival advantage over cells with persisting DSBs so that cells with TD are selected for in the assay. If this were true and if many cells in mu2 or mus304 were unable to establish telomere at D4A and later died, preferential recovery of the uncut homolog and impaired male fertility due to germ cell loss would have been observed. Neither was observed for any of the mutants that were tested suggesting that apoptosis is not a normal response in these cells defective for damage response or repair. The proposition is supported that defective checkpoints lead to persistence of DSBs allowing more time for telomere establishment (Beaucher, 2012).
The hypomorphic tefu and nbs mutations enhanced TD recovery similarly but to a lesser extent than mu2 and mus304 null mutations. It is possible that the underlying mechanism, i.e. persisting DSBs due to defective checkpoints, is common for both groups of mutants. However, null mutations were used and it was shown that the checkpoint functions of ATM and, to some degrees, MRN are less prominent in Drosophila than the ones controlled by ATR and ATRIP. It is speculated that other functions of ATM and MRN might help inhibit de novo telomere formation. In particular, ATM and MRN are essential for end tethering during DSB repair. It is imaginef that the two ends of the DSB induced at D4A are allowed to separate in great distance in tefu or nbs cells, which would impede repair giving more time for telomere formation. In addition, the MRN complex is important for both HR and NHEJ repair of DSBs. The nbs mutation might affect telomere formation via its function in DSB repair as the results suggest that inhibiting DSB repair facilitates telomere formation (Beaucher, 2012).
Two modes of repair of the DSB at D4A will recreate the I-SceI cut site: precise end joining and recombination with the sister chromatid, making the chromosome susceptible to a second round of cutting. Therefore, any events leading to the disruption of the cut site would be favored in the presence of continuous I-SceI expression. de novo telomere formation represents one of these events. Consistently, when HR was impaired by the spnA mutation or end joining by Lig4, TD recovery rate increased. Loss of SpnA has a larger effect than loss of Lig4, which is consistent with a previous observation that inter-sister HR is the predominant pathway for the repair of I-SceI induced DSBs in the male germline. Therefore, channeling of DSBs is likely the cause for elevated TD recovery in repair defective germline (Beaucher, 2012).
In the assay, scoring progeny that inherited a TD but have lost white sequences helps illustrate the extent of nucleolytic degradation of chromosome ends before and after de novo telomere formation. It is surmised that a longer half-life of DSB in checkpoint mutants would result in more extensive degradation. Consistently, mutants with suspected defects in checkpoint functions (mu2, mus304, tefu and nbs) all led to increased white-loss in TD progeny. However, in tefu and, to a lesser extent, nbs mutants, this increase is disproportionally larger than the increase in total TD recovery. For example, close to half of TD progeny from tefu suffered a loss of white expression. These results suggest that ATM and NBS normally inhibit end degradation at telomere ends. Contrary to mutants that prolong the presence of DSBs, it is considered that mutants defective in individual DSB repair pathways are unlikely to cause extensive end-degradation before telomere formation. Different repair pathways compete for the available DSBs so that when one is defective DSBs are efficiently repaired by others. This suggests that defects in a single repair pathway is unlikely to prolong the presence of DSBs. Consistently, the increases in TD recovery in spnA and Lig4 germline were not accompanied by significantly elevated levels of white-loss (Beaucher, 2012).
The Inverted repeat-binding protein (Irbp) mutant is interesting in that it behaved differently from any other mutants in the assay, causing a dramatic increase of white-loss TD events but without a significant increase in the overall TD recovery. Several points concerning Ku70's function are deduced from these results. First, loss of Ku70 does not impact precise end joining to a degree similar to the Lig4 mutation. Second, imprecise NHEJ is infrequently used for DSB repair at D4A in the male germline so that its disruption by the loss of Ku70 does not lead to significant channeling of DSBs for telomere formation. Thirdly, the excessive end-degradation in Irbp germ cells is likely specific to telomeric ends, and occurs after the commitment of the DSB to a telomeric fate but before the establishment of a functional telomere. This last point was based on the observation that events with white-loss followed by successful NHEJ) were not recovered at a higher frequency in the mutant background. It is also shown that once a functional telomere has been established at D4A, loss of Ku70 has no effect on the rate of end attrition. It is speculated that once a DSB is committed to a telomeric fate, the binding of Ku70 prevents excessive nucleotytic attrition before the functional establishment of a protective cap. Intriguingly, Ku70 seems to have no role in either fate determination of DSB ends or cap establishment on ends (Beaucher, 2012).
Remarkably, a close to 20-fold increase was observed in new telomere formation accompanied by a transposon attachment event in mus304 germ cells. This frequency is likely to be an underestimate due to the fact that transposon attachment to D4A end that has lost part of white could not be identified in the assay that was used (Beaucher, 2012).
In yeast S. ceravisiae, the Mec1/ATR kinase, and presumably its partner ATRIP, prevents accumulation of the Cdc13 protein at DSBs. Cdc13 is a member of the Cdc13-Stn1-Ten1 (CST) telomeric complex essential for telomere protection and the recruitment of telomerase activities to telomeres. Interestingly, the Drosophila Verrocchio (Ver) protein was recently identified as an essential capping protein and shares limited homology with Stn1 proteins from other organisms. This suggests that a similar CST complex might exist in Drosophila. It is speculated that Drosophila CST might accumulate at DSBs in the absence of Mus304/ATRIP, leading to more efficient recruitment of the transposon machinery, similar to CST's role in telomerase recruitment in the other systems (Beaucher, 2012).
Ku70 heterozygosity has been shown to lead to elevated rates of transposon attachment in the female germline. This increase happens over a few successive generations. No evidence was observed of rampant transposon attachment to D4ATD from Southern blot analyses on TDs that have been kept in the Irbp background for several generations. However, the crossing scheme only allowed TDs to be present in the mutant germline from males (Beaucher, 2012).
It was not surprising that almost all mutations tested in this study lead to increases in the recovery of events associated with de novo telomere formation. It is consistent with the idea that telomere formation might be a backup mechanism to all modes of damage repair and response in germ cells. Although the candidate approach in identifying factors essential for telomere establishment is far from comprehensive, a picture has emerged in which factors responsible for the recruitment and execution of damage repair and response activities are also responsible for inhibiting telomere formation. In the absence of these activities, the DNA and chromatin structures at the ends might be sufficient for the recruitment of the protective cap. If this were true, only defects in the protective cap itself would have a negative effect on telomere establishment on DSBs. This hypothesis was not tested due to the lack of hypomorphic mutations in capping components (Beaucher, 2012).
The role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51 was investigated. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. These findings indicate that Rad54 and Rad51 have evolved to function with chromatin, the natural substrate, rather than with naked DNA (Alexiadis, 2002).
Homologous recombination occurs in the repair of DNA double-strand breaks as well as during meiosis. Genetic studies in Saccharomyces cerevisiae led to the identification of the RAD52 epistasis group of genes (which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2) as components of the recombinational repair pathway. These genes are conserved from yeast to humans. A central protein in this pathway is Rad51, which is related to the bacterial RecA protein. Both Rad51 and RecA are able to mediate strand invasion and annealing to yield a D loop, which is a key step in the recombination process. In this reaction, Rad51 (or RecA) forms a nucleoprotein filament on single-stranded DNA in the presence of ATP, and this filament is used for homologous pairing with a double-stranded DNA molecule. The efficiency of strand pairing by Rad51 (between single-stranded DNA and homologous duplex DNA) has been shown to be stimulated by the presence of additional factors such as RP-A, the Rad55-Rad57 heterodimer, Rad52, and Rad54 (Alexiadis, 2002 and references therein).
To study homologous recombination in the context of chromatin, focus was placed on the ability of purified recombinant Rad51 and Rad54 to catalyze D-loop formation between single-stranded DNA and homologous double-stranded DNA that is packaged into chromatin. The function of Rad54 in chromatin is of particular interest because it is a member of the Snf2-like family of ATPases. The Snf2-like family includes proteins such as Swi2/Snf2, Sth1, ISWI, Ino80, and Mi-2/CHD3/CHD4, which are the ATPase subunits of chromatin-remodeling factors that catalyze the mobilization of nucleosomes. It thus seemed possible that Rad54 would be important for homologous recombination in chromatin. Therefore, whether purified Rad51 and Rad54 can mediate D-loop formation with chromatin was investigated (Alexiadis, 2002).
To study the biochemical properties of Rad51 and Rad54, focus was placed Drosophila Rad51 and Rad54 (with C-terminal Flag tags) in Sf9 cells by using a baculovirus expression system, and then the proteins were purified to near homogeneity by FLAG immunoaffinity chromatography. The ability of these factors to mediate D-loop formation between a radiolabeled, single-stranded oligonucleotide (termed DL2; 135 nt) and a homologous, double-stranded plasmid (pU6LNS; 3291 bp) was tested. In this reaction, Rad51 assembles onto the single-stranded oligonucleotide in the presence of ATP to give a nucleoprotein filament, and then Rad54 interacts with the Rad51-oligonucleotide complex and facilitates the strand-pairing reaction. These experiments reveal that purified recombinant Drosophila Rad51 and Rad54 can catalyze the formation of D loops in a manner that is dependent on Rad51, Rad54, ATP, and homologous plasmid DNA (Alexiadis, 2002).
Next, the ability of Rad51 and Rad54 to catalyze D-loop formation in chromatin was tested. In these experiments, chromatin was reconstituted by salt dialysis techniques. The salt dialysis chromatin (SD chromatin) was prepared by gradually decreasing the salt concentration in a mixture of plasmid DNA and purified core histones from Drosophila embryos, and fully reconstituted chromatin was separated from partially reconstituted chromatin by sucrose gradient sedimentation. Micrococcal nuclease digestion analysis of the chromatin samples revealed that the salt dialysis chromatin consisted of closely packed arrays of nucleosomes. D-loop reactions were performed with the SD chromatin. These experiments revealed that Rad51 and Rad54 are able to form D loops with SD chromatin at an efficiency that is slightly higher than that obtained with naked DNA. Moreover, the rate of D-loop formation by Rad51 and Rad54 with chromatin is similar to that seen with naked DNA. In contrast, the Escherichia coli RecA protein is able to mediate D-loop formation with naked DNA, but not with chromatin. Thus, these experiments, which were performed with completely purified components, show that Rad51 in cooperation with Rad54 can mediate D-loop formation with chromatin with comparable efficiency and kinetics as with DNA, whereas the bacterial recombinase RecA is unable to mediate strand pairing with chromatin. The inability of RecA to function with chromatin is consistent with previous studies carried out with mononucleosomes, and further suggests that RecA is lacking a chromatin-specific function that is present in Rad51 and/or Rad54. In this regard, whether Rad54 could stimulate D-loop formation in chromatin by RecA was tested, but no activity was demonstrated (Alexiadis, 2002).
The bulk of the eukaryotic genome appears to possess little superhelical tension, and therefore the effect of torsional stress upon D-loop formation by Rad51 and Rad54 was investigated. To this end, the salt dialysis chromatin was relaxed with purified topoisomerase I. The salt dialysis chromatin was reconstituted by using supercoiled plasmid DNA in the absence of topoisomerases. Under these conditions, the DNA remains chemically unchanged, since no phosphodiester bonds are broken. Hence, in the absence of topoisomerase I, the numbers of supercoils in the naked DNA and chromatin (which was deproteinized prior to electrophoresis) are essentially identical. When topoisomerase I is added to the chromatin, the unconstrained supercoils are relaxed, but upon deproteinization, the resulting DNA exhibits supercoils that are caused by the wrapping of the DNA in nucleosomes, because the wrapping of the DNA around each histone octamer constrains approximately one negative supercoil (Alexiadis, 2002).
Strand-pairing reactions were performed with DNA and salt dialysis chromatin in the absence or presence of topoisomerase I. With naked DNA, a >100-fold reduction in the efficiency of D-loop formation was observed upon relaxation of the template with topoisomerase I. Notably, this >100-fold decrease in strand-pairing efficiency is much more pronounced than the twofold reduction seen with yeast Rad51, Rad54, and RPA. This difference could potentially be due to the use of yeast versus Drosophila factors, the presence or absence of RPA, the length of the single-stranded DNA (5386 nt, Van Komen, 2000; 135 nt, this study), and/or the concentration of Rad51 in the reaction medium (1500 nM, Van Komen, 2000; 200 nM, this study). Note, however, that stimulation of D-loop formation by purified RPA was not observed in reactions performed in this study. In contrast to the effects seen with naked DNA, relaxation of the chromatin by topoisomerase I has little effect on the efficiency of D-loop formation by Rad51 and Rad54. Thus, in the absence of superhelical tension, strand pairing by Rad51 and Rad54 occurs with higher efficiency in chromatin than in naked DNA (Alexiadis, 2002).
Because Rad54 is related to the ATPase subunit of chromatin-remodeling complexes, whether Rad54 possesses chromatin-remodeling activity was tested. The ability of Rad54 and/or Rad51 to facilitate the access of a restriction enzyme (HaeIII) to DNA packaged into nucleosome arrays was tested. ACF was used as a positive control. This type of restriction-enzyme accessibility assay has been used for the analysis of chromatin remodeling in vivo, the biochemical purification of the CHRAC chromatin-remodeling factor, the characterization of the INO80.com remodeling complex, and the comparative analysis of six chromatin-remodeling complexes (ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, dNURF). Neither Rad54 alone nor Rad51 alone exhibits any detectable chromatin-remodeling activity in the absence or presence of the DL2 oligonucleotide. In sharp contrast, Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. The ability of Rad54 and Rad51 to rearrange chromatin structure is consistent with their ability to catalyze strand pairing with chromatin. It is also notable that Rad54 requires the presence of Rad51 to function as a chromatin-remodeling factor (Alexiadis, 2002).
In conclusion, these studies have revealed that D-loop formation by Rad54 and Rad51 occurs with >100-fold higher efficiency with chromatin relative to naked DNA in the absence of superhelical torsion. In addition, Rad54 and Rad51 act cooperatively in the ATP-dependent remodeling of chromatin. This ability of Rad54 and Rad51 to alter chromatin structure is likely to be related to their chromatin-specific function in the strand-pairing reaction. These findings provide an example of optimized function of eukaryotic DNA-using proteins in chromatin. Moreover, it is possible that the use of chromatin templates, instead of naked DNA templates, might similarly increase the efficiency of targeted homologous recombination in vivo (Alexiadis, 2002).
Biochemical studies of the BLM gene product have shown its ability in conjunction with topoisomerase IIIalpha to resolve double Holliday structures through a process called 'dissolution.' This process could prevent crossing over during repair of double-strand breaks. This study describes an analysis of the Drosophila BLM gene, DmBlm, in the repair of double-strand breaks in the premeiotic germ line of Drosophila males. With a repair reporter construct, Rr3, and other genetic tools, it is shown that DmBlm mutants are defective for homologous repair but show a compensating increase in single-strand annealing. Increases of 40- to 50-fold in crossing over and flanking deletions also were seen. Perhaps most significantly, the template used for homologous repair in DmBlm mutants is itself subject to deletions and complex rearrangements. These template disruptions are indicative of failure to resolve double Holliday junctions. These findings, along with the demonstration that a weak allele of topoisomerase IIIalpha has some of the same defects as DmBlm, support the dissolution model. Finally, an analysis of DmBlm mutants in conjunction with mus81 or spnA (Rad51) reveals a second function of BLM distinct from the repair of induced double-strand breaks and possibly related to maintenance of replication forks (Johnson-schlitz, 2006).
The human BRCA2 cancer susceptibility protein functions in double-strand DNA break repair by homologous recombination and this pathway is conserved in the fly Drosophila. Although a potential Drosophila BRCA2 orthologue (Brca2; CG30169) has been identified by sequence similarity, no functional data addressing the role of this protein in DNA repair is available. This study demonstrates that depletion of Brca2 from Drosophila cells induces sensitivity to DNA damage induced by irradiation or treatment with hydroxyurea. Brca2 physically interacts with rad51 (spnA), and the two proteins become recruited to nuclear foci after DNA damage. A functional assay for DNA repair demonstrated that in flies Brca2 plays a role in double-strand break repair by gene conversion. Finally, it was shown that depletion of Brca2 in cells is synthetically lethal with deficiency in other DNA repair proteins including parp. The conservation of the function of BRCA2 in Drosophila will allow the analysis of this key DNA repair protein in a genetically tractable organism potentially illuminating mechanisms of carcinogenesis and aiding the development of therapeutic agents (Brough, 2008).
The Drosophila genome carries a potential BRCA2 orthologue as indicated by the presence of BRC (RAD51-binding) motif sequences (Lo, 2003). However, this protein does not contain recognisable DNA and DSS1 binding domains, both characteristics of the mammalian BRCA2 protein. Using both cell culture and whole organism genetic approaches this study has shown that despite lacking these motifs the CG30169 allele is the likely functional BRCA2 orthologue. Using Drosophila cells in culture it was shown that a deficiency for the Brca2 protein induces sensitivity to both X-rays and the DNA-damaging drug HU. This phenotype is typical of eukaryotic cells deficient in DNA repair and has been demonstrated using various DNA damaging agents in the fly for a number of mutant genes, including rad51. By comparison, only a few studies have shown a heightened sensitivity to DNA damage in fly cells in culture (Brough, 2008).
I-SceI-based assays were subsequently performed to investigate the role of Drosophila BRCA2 in DSBR. The results clearly showed that Brca2 is essential at least for inter-homolog gene conversion repair. In this respect, the Brca2 mutant behaves similarly to Drosophila rad51 and rad54 mutations. These results are also consistent with those from mammalian and fungal studies. Therefore, it is concluded that the essential function of Brca2 in homology-directed DSBR is evolutionarily conserved despite poor conservation in protein sequence (Brough, 2008).
Further evidence that Brca2 is involved in DNA repair was provided by investigating a possible interaction between Brca2 and Rad51. Co-immunoprecipitation showed that the two proteins interact in both the presence and absence of DNA damage. However, using immunofluorescence analysis it was demonstrated that the proteins co-localise within nuclear foci following DNA damage but not before and that Brca2 is likely to be involved in the recruitment of Rad51 to the sites of damage. The interaction of Brca2 and Rad51 is consistent with the presence of three BRC repeats within Brca2. However, Brca2 unlike other BRCA2 orthologues lacks a recognisable OB fold domain capable of binding DNA. It seems possible Brca2 interacts with another protein which performs this function (Brough, 2008).
Heterozygous germline mutations of the BRCA2 gene in humans confer a high risk to a range of cancers. The mechanism for this is through genome instability caused by loss of the wild-type BRCA2 allele in tumours. One approach to the development of new therapeutic approaches is to target the deficiency in DNA repair. Such synthetic lethal therapeutics are in development via the inhibition of the enzyme PARP which is involved in base excision repair. To extend this approach it is important to identify additional synthetic lethal interactions. Drosophila cells have already been used to identify evolutionarily conserved pathways and genetic interactions. Therefore, to test the feasibility of such an approach in DNA repair pathways the synthetic lethal interaction of Brca2 deficiency with a number of DNA repair genes was studied (Brough, 2008).
This study shows that synthetic lethal interactions exist between Brca2 and Parp, analogous to the mammalian system. This suggests that the interactions between DNA repair pathways are evolutionarily conserved. In addition, an interaction between alternative dsDNA break repair pathways (NHEJ) was observed. Similar synergy between the HR and NHEJ pathway has already been observed in Drosophila; for instance, crossing Blm or LigIV mutant flies with Rad54 mutant flies was shown to increase the sensitivity of the resulting progeny to DNA damage. The functional conservation of BRCA2 as well as the conserved interplay of HR with other DNA repair pathways, as demonstrated by synthetic lethal interactions, suggests that Drosophila will be a powerful system for dissecting BRCA2 biology as well as aiding the development of new therapeutic approaches (Brough, 2008).
Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination (see Hypothetical model for BRCA2 function in HR, Venikataram, 2002). Much about its precise function in the DNA damage responses is, however, not yet known. Null mutations have been made in the Drosophila homolog of BRCA2, and the levels of homologous recombination, non-homologous end-joining, and single-strand annealing were measured in the pre-meiotic germline of Drosophila males. Repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. During meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal (Klovstad, 2008).
The genomic stability of eukaryotic cells is constantly challenged by exogenous and endogenous stresses that can lead to the loss or alteration of genetic material. Genomic stability is maintained through robust DNA repair and checkpoint pathways that are tightly coordinated with each other and the developmental cell cycle progression of the organism. For example, mutations in meiotic DNA repair enzymes in Drosophila cause defects in the cell cycle and developmental progression of the egg due to a failure to repair meiotic recombination intermediates. The tumor suppressor and breast cancer susceptibility gene, BRCA2, has been implicated in playing a central role in maintaining genomic stability, but the extent to which BRCA2 is involved the coordination of DNA repair, checkpoints, and developmental progression remains to be determined (Klovstad, 2008).
Murine cells depleted for BRCA2 spontaneously accumulate broken chromosomes and chromatids, triradial and quadriradial structures, and gross chromosomal rearrangements (Yu, 2000; Patel, 1998). A key function of BRCA2 is the regulation of the Rad51 recombinase during DNA repair by homologous recombination (HR). During HR, Rad51 assembles into a nucleoprotein filament with single-stranded DNA at the site of a double-stranded break (DSB) in order to initiate strand invasion of the homologous chromosome (Paques, 1998). Recent structural studies have illuminated how BRCA2 regulates Rad51 (Yang, 2005; Davies, 2007; Esashi, 2007). BRCA2 contains two regions that mediate binding to Rad51: a stretch of 8 repeated short motifs termed the BRC repeats and a C-terminal region termed TR2. The BRC repeats bind the Rad51 oligermerization domain to disrupt Rad51 self-oligermerization. BRCA2 then catalyzes the formation of the nucleoprotein filament at the single-stranded/double-stranded DNA junction flanking a DSB. This filament is stabilized in a cell-cycle-dependent manner by the TR2 domain of BRCA2. The role of BRCA2 in homologous recombination is likely critical for its role as a tumor suppressor, but BRCA2 is a large protein with many binding partners and it is likely to play multiple roles in safeguarding genomic stability (Klovstad, 2008).
Several requirements for BRCA2 outside of homologous recombination have been suggested by protein interaction and cell culture studies, but these functions are far less understood. Most notably, BRCA2 has been implicated in two S-phase checkpoints: the intra-S phase checkpoint and the replication checkpoint. During the intra-S phase checkpoint irradiation-induced lesions outside of the replication fork cause partial depression of replication. The replication checkpoint stabilizes replication forks and decreases replication levels in response to lesions at the replication fork. Requirements for BRCA2 in replication fork stabilization after hydroxyurea treatment and in suppressing radioresistant replication have also been described (Kraakman-van der Zwet, 2002; Lomonosov, 2003). The G1/S, S/M, and G2/M checkpoints have been found to be largely intact in BRCA2 mutants. However, these studies have used hypomorphic mutations that preserve half or more of the N-terminal region of BRCA2 due to proliferative defects of BRCA2 null mutants. As modest to severe increases in breast cancer susceptibility have been associated with mutations in other checkpoint genes, further elucidation of the mechanism of the role of BRCA2 in checkpoints is needed (Klovstad, 2008).
Drosophila has emerged as a useful model for studying DNA repair and checkpoint control of genome stability. The DNA repair and checkpoint pathways are remarkably well conserved between flies and higher organisms (Sekelsky, 2002). Notably, though the function of many of these genes is well conserved, null mutants in Drosophila are sometimes viable in cases in which null mutations in higher organisms result in lethality and complicate mammalian developmental studies. In this study null mutations have been made in the Drosophila homolog of BRCA2 (CG30169), and show that unlike in mammals, Drosophila brca2 null mutants are viable. Detailed descriptions are presented of DSB repair pathway balance and irradiation-induced checkpoint function in animals genetically null for brca2. CG30169 represents a functional BRCA2 homolog required for DSB repair in mitotic and meiotic tissues. Additionally, a novel role was uncovered for brca2 in the meiotic recombination checkpoint. Finally it was shown that Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a mechanism for the role of Brca2 in checkpoint control (Klovstad, 2008).
These results demonstrate that the predicted gene CG31069 is a functional BRCA2 homolog required for meiotic and mitotic homologous recombination. Null mutations were made in Drosophila brca2 in two distinct genetic backgrounds, and the ovarian defects were rescued by genomic rescue. Unlike in mammals in which null mutations are early embryonic lethal (Ludwig, 1997), Drosophila brca2 null mutants are homozygous viable, possibly because of the long period of maternal gene expression during Drosophila embryonic development. It is still uncertain whether Drosophila Brca2 contains a cryptic DNA binding domain similar to mammalian BRCA2 or if this function is encoded in a tightly regulated interacting protein. Further biochemical studies will be necessary to resolve this question, but it is clear from functional analysis of the role of Brca2 in DNA repair, as well as a recently reported physical interaction with SpnA/Rad51 in Drosophila (Brough, 2008), that Drosophila brca2 represents a functional homolog of the human breast cancer susceptibility gene. Clearly, due to the viability of Drosophila brca2 null mutants and the power of Drosophila genetics, Drosophila offer a promising new opportunity for uncovering novel roles for BRCA2 during development. This work presents a thorough characterization of the role of Brca2 in DSB repair and a novel function for Brca2 in the meiotic recombination checkpoint was uncovered (Klovstad, 2008).
Using the Rr3 assay (Preston, 2006), which monitors the repair of a DSB at an I-SceI endonuclease site flanked by partial copies of the reporter dsRed, this study showed that in brca2 mutants, DSB repair is shifted towards repair by potentially mutagenic repair pathways. Repair by homologous recombination is dramatically decreased in brca2 mutants, and repair by single-strand annealing predominates. Repair by single strand annealing (SSA) always results in the loss of the sequences between annealed repeats. SSA repair is restricted to DSBs flanked by repetitive elements, though due to the highly repetitive nature of higher eukaryotic chromosomes, SSA repair can represent a significant source of mutagenesis in higher eukaryotes. The current results contrast with studies in C. elegans, in which indirect in vivo experiments and in vitro annealing experiments (Martin, 2005) have lead to the suggestion that the C. elegans BRCA2 homolog is required for SSA repair. The current results are more similar to the effects seen in mammalian cell culture experiments with hypomorphic BRCA2 mutations, in which decreases in HR repair correlated with increases in SSA repair (Larminat, 2002; Stark, 2004). In addition, Brough (2008) also has reported a similar inverse relationship between SSA and HR-h in a Drosophila brca2 mutant using a simplified DSB repair assay (Klovstad, 2008).
In the Rr3 assay when one pathway is compromised, the sum of the relative pathways usage in these mutants still equals near 100%, even though the percentages are calculated from different populations and are not forced to equal 100%. This observation plus the fact that different effects on repair pathway balance have been observed among mutants with decreases in the same pathway suggest that regulated compensation can occur. For example, mutations in mus101 and mei-41 both result in a decrease in SSA, but the former are compensated by increases in non-homologous end joining (NHEJ) and homologous recombination using the homologous chromosome (HR-h) while the latter is compensated by NHEJ only. In cross 2, in which the EJ1 chromosome was present and HR-h pathway was available, a significant increase was seen in the use of the SSA, but no significant difference in the relative level of NHEJ in brca2 mutants, indicating compensation by the SSA pathway in brca2 mutants (Klovstad, 2008).
Compensation of decreases in HR-h by the SSA pathway seems to be a common response to deficiencies in genes required for HR-h. In mammalian cell culture studies, Stark (2004) found a similar inverse relationship between SSA and HR in Brca2 and Rad51 mutants, while Brca1 mutants had decreases in both SSA and HR. Using the Rr3 assay, Johnson-Schlitz (2007) found that the significant decreases in HR-h in Drosophila dmBlm, top3α, and spnA mutants were compensated entirely through increases in SSA, while in okra mutants compensation occurred through significant increases in both SSA and NHEJ. In the current experiments compensation in okra mutants was observed entirely through SSA, and the exact cause of the discrepancy is unclear. Possibly the discrepancy lies in the use of different endonuclease sources or in different combinations of okra alleles used (okraAA/RU versus okraRU/WS), though mutagen sensitivity studies have suggested that the AA and WS alleles are of similar strength. Regardless, it is clear that brca2 mutants are compensated by SSA, similar to most mutants deficient for HR-h in Drosophila and other organisms (Klovstad, 2008).
It is also notable that short-tract HR-h is more strongly affected in brca2 mutants than long-tract HR-h. Short-tract HR-h was decreased 64-fold relative to wild-type values and long-tract HR-h was decreased 9-fold. The residual HR-h repair probably reflects repair that occurred early when low levels of maternal Brca2 were still present and wild-type levels of Brca2 may be required to restrict the extent of gene conversion during HR-h repair. Gene conversion tract length during HR-h repair has important implications in maintaining genomic integrity as the potential for loss of heterozygosity increases with increasing tract length. Mutations in okra led to a similar shift towards long-tract HR-h, and homozygous and heterozygous mutations in spnA have also been observed to alter the balance of HR-h in towards long-tract HR-h (Johnson-Schlitz, 2007). These results suggest that reductions in levels of the enzymes required for strand invasion can result in increased rates of loss of heterozygosity during HR-h repair. Increased long-tract HR-h repair relative to short-tract HR-h repair in brca2 mutants may represent increased rate of DNA synthesis, increased resection prior to strand invasion, increased stability of recombination intermediates, unequal repair of the heteroduplex DNA, or a combination of these processes. In mammalian cells, an increase in the extent of gene conversion has been observed in both Rad51K133R and Xrcc3, a Rad51 paralog, mutants; though these observed increases are thought to have arisen from different mechanisms due to differences seen in gene conversion tract continuity. Models explaining the increase in the inclusion of the 16 bp deletion in Drosophila brca2 and spnA mutants would first need to determine whether conversion tracts in these mutants are continuous or discontinuous by using a more complicated reporter design (Klovstad, 2008).
This study found a novel requirement for brca2 in transduction of the meiotic recombination checkpoint signal. Initially the meiotic phenotypes of brca2 mutants were surprisingly different from the spindle class mutants previously studied. First, even with the exacerbation of the ventralization defect by growth at 25 °C, the eggshell ventralization phenotype of brca2 mutants was significantly weaker in spite of similar levels of persistent DSBs. Second, the kinetics of the eggshell phenotype were opposite to the kinetics of classical spindle mutants. In spnA,B,C,D and okra mutants the phenotype is weak during the initial days in which the females are fed yeast, but after 5-7 days on yeast spindle mutants lay predominately severely ventralized eggs. In brca2 mutants, ventralized eggs were only reliably laid during the first 1-4 days on yeast. Given these results, it is now clear that the requirement for brca2 in efficient transduction of the checkpoint signal masks the strong eggshell ventralization phenotype that is normally suggestive of a role in meiotic DSB repair. As an increasing number of proteins with dual roles in DNA repair and checkpoint function are being identified, it will be interesting to see if there are additional dual function proteins functioning in Drosophila meiosis. While the classical meiotic repair mutants have strong oogenesis phenotypes, weak or absent eggshell patterning defects may not preclude a role in meiotic DNA repair if coupled to a role in checkpoint transduction (Klovstad, 2008).
The ovarian phenotypes of brca2 mutants were suppressed by mutations in chk2, but were not suppressed by mei-41 mutations. This finding is in contrast to the other spindle class mutants which are suppressed by both mei-41 and chk2 mutations. It is proposed that in females with an intact checkpoint response, the checkpoint activation is dependent upon mei-41 and chk2. However, because Brca2 acts in a similar step in the checkpoint pathway as Mei-41, no additional suppression is observed in the mei-41; brca2 double mutant. Since both the eggshell and karyosome defects of brca2 mutants can be suppressed by chk2 it is probable that the residual checkpoint activation in brca2 mutants is due to activation of Chk2 by the upstream checkpoint kinase Atm. It is not currently possible to test the involvement of Atm in the checkpoint, since even viable, hypomorphic atm single mutants have eggshell and karyosome defects. There is however evidence that Mei41-independent checkpoints exist in Drosophila meiosis. Upregulation of transposable elements in the Drosophila germline, as seen in cutoff mutants, results in eggshell patterning defects that can be suppressed by chk2 but not by mei-41 mutations (Chen, 2007). The checkpoint activated in cutoff mutants is, however, distinct in at least some aspects from the checkpoint activated in brca2 mutants. Unlike in brca2 mutants, the checkpoint activated in cutoff mutants results in a loss of germline cells as well as eggshell patterning defects, and these defects cannot be suppressed by mutations that prevent DSB formation (Klovstad, 2008).
According this model, Mei41-independent checkpoint activation in brca2 mutants is strong enough, with respect to either signal strength or signal duration, to result in a karyosome defect but not strong enough to result in strong eggshell patterning defects. Clearly Mei-41 is responsible for the bulk of checkpoint activation in classical spindle mutants but it remains possible that Atm may play a supporting role. It is notable that the classical spindle phenotypes are typically scored after 5-7 days on yeast at ambient temperature and that at these conditions the brca2 single mutant phenotype was very weak. Therefore it is possible that under the conditions used in this study mei-41 mutations may also not completely suppress the classical spindle mutations. In interpreting the role of brca2 in the transduction of the meiotic recombination signal, focus was placed on the eggshell phenotype since brca2 single mutants have karyosome defects. This phenotype is similar to that seen in hus1 mutant females. hus1 mutants are able to suppress the eggshell defects of spindle mutants but do not suppress the karyosome defects of the spindle mutants as hus1 single mutants have karyosome defects (Abdu, 2007). Though it has not been tested in this study, it seems possible that, similar to brca2 mutants, the hus1 mutant karyosome defect is a result of persistent DSBs and partial activation of the checkpoint (Klovstad, 2008).
It was also found that Brca2 co-immunoprecipitates with Rad9. The 9-1-1 complex forms a heterotrimeric ring that is loaded onto resected single-stranded DNA flanking a DSB following replication stress. The 9-1-1 complex associates with DSBs independent of ATR, and mediates Chk1 phosphorylation by ATR through interaction with TopBP (Delacroix, 2007). The 9-1-1 complex may or may not use a similar mechanism to activate Chk2 during the meiotic recombination checkpoint. Although the precise functional relevance of the Rad9-Brca2 interaction remains to be explored, co-immunoprecipitation results further suggest that the role of Brca2 in checkpoint control is upstream of Chk1/Chk2 (Klovstad, 2008).
Research in mammals and work in Drosophila has shown that the 9-1-1 complex and BRCA2 are specifically required for the checkpoints thought to be activated in response to large stretches of single-stranded DNA. In Drosophila they are required for the meiotic recombination checkpoint, but not the irradiation checkpoints. In mammals, BRCA2 and the 9-1-1 complex are required for several S-phase checkpoints. Now that a physical connection between Rad9 and Brca2 has been observed, it will be interesting to determine the degree of functional overlap between Brca2 and the 9-1-1 complex in the DNA damage responses. In light of the absence of a predicted DNA binding domain in Drosophila brca2 it is tempting to predict that the Drosophila 9-1-1 complex and Brca2 may have a common role in both repair and checkpoints. While the functional overlap is complete for the checkpoints examined to date, it is clear from mutagen sensitivity assays that the functional overlap between 9-1-1 and Brca2 in DNA repair is not absolute. hus1 mutants are severely sensitive to MMS, but not to IR, while brca2 mutants are moderately sensitive to MMS and severely sensitive to IR (Abdu, 2007; Klovstad, 2008).
In conclusion the Drosophila homolog of BRCA2 is required for mitotic and meiotic homologous recombination and in the absence of brca2 error-prone repair predominates. brca2, similar to the 9-1-1 complex with which it physically interacts, has a second requirement during meiosis in the activation of the meiotic recombination checkpoint but is not required for checkpoints that respond to irradiation induced damage, indicating a specialized role for brca2 in checkpoint control (Klovstad, 2008).
RNA blotting analysis detects a single Rad51 transcript of about 1.35 kb that is present throughout development at low levels. Transcript levels are induced at least tenfold in ovaries, as measured by RNase protection analysis, suggestive of a role in female meiosis. Transcript levels are significantly lower in testes than in bulk RNA of adult males, however, indicating that Rad51 may be repressed in meiosis of Drosophila males (McKee, 1996).
Twenty-two new alleles of spindle-A have been identified by mutagenesis screening. All new spnA alleles are viable in trans to the original spnA alleles (spnA003, spnA050 and spnA057) and in trans to a deficiency for the region, suggesting that they are not affecting a function essential for viability. Like the original spnA alleles, the new alleles show ~100% maternal-effect embryonic lethality. All mutant lines produce a spectrum of eggshell ventralization phenotypes similar to those described for known mutations affecting the Egfr signaling pathway, ranging from fused dorsal appendages to complete ventralization (Gonzalez-Reyes, 1997). For most spindle-class mutants, the eggshell phenotype has been attributed to a defect in RNA and protein localization or protein synthesis of the Egfr ligand Gurken (Grk) (Gonzalez-Reyes, 1997). In spnA mutants, the level and distribution of Grk protein is disrupted (Gonzalez-Reyes, 1997). In one example, instead of the normal crescent of Grk protein along the dorsal-anterior side of the wild-type oocyte nucleus facing the somatic follicle cells, Grk protein distribution is less coherent and only found in a few spots along the mutant oocyte nucleus. Another common feature shared among the spindle-class mutants is a disruption in oocyte nuclear morphology (Gonzalez-Reyes, 1997; Ghabrial, 1998). Mature wild-type oocytes contain highly compact chromatin called the karyosome. In spnA mutant oocytes, as well as in other spindle-class mutants, the DNA is less organized and diffuse. In contrast to the wild type, where DNA is found as a condensed sphere in the center of the nucleus, DNA in spnA mutant oocytes clusters along the periphery of the nucleus adjacent to the nuclear membrane (Staeva-Vieira, 2003).
spnA was mapped to the cytological region 99D01-99E01. The Berkeley Drosophila Genome Project predicted a Rad51-like gene (CG7948) to reside within this region of the genome. CG7948 shows strong sequence similarity to the yeast and mammalian Rad51 gene. Since two other spindle-class genes, spnB and okra, encode members of the Drosophila Rad51 family and Rad54, respectively (Ghabrial, 1998), CG7948 was a likely candidate for spnA. Sequence analysis of spnA alleles revealed unique missense mutations within CG7948. Thus, spindle-A encodes a Drosophila Rad51-like gene (Staeva-Vieira, 2003).
The molecular characterization of all 25 spnA alleles revealed 22 missense mutations (spnA009A and spnA032B contained the same mutation) and two stop codon mutations. Each missense mutation affects an amino acid conserved from yeast to human Rad51. Western analysis using an antibody that was raised against the entire Drosophila Rad51 protein revealed that spnA093A, which has an early stop codon at amino acid 70, produces no detectable protein and classifies as a protein null. The other nonsense allele spnA048B introduces a late stop at amino acid 265 and produces a truncated protein. Three missense alleles, spnA087A and spnA010A, which have missense mutations in the Walker-A box and Walker-B box, respectively, and spnA057, which has a change within the DmRad51 core domain, were also analyzed. Western analysis revealed that the missense alleles produce stable proteins that are of similar size to the wild-type protein. Since all alleles of spnA including the null allele spnA093A are viable in trans to the deficiency, Df(3R)X3F, it is concluded that SpnA function is required for oogenesis but is not essential for normal cell viability (Staeva-Vieira, 2003).
Since spnA and spnB are expressed in the soma as well as the germline, it was of interest to investigate a possible somatic function for these genes. To determine if these proteins function in the mitotically active cells of the soma, spnA, spnB and spnB;spnA doubly mutant embryos and larvae were exposed to 20 Gy of ionizing radiation (X-rays) and examined for survival. The progeny of heterozygous parents were irradiated at either 0-24 h, 24-48 h or 48-72 h after egg laying (AEL). The survival of the irradiated progeny was compared to that of their unirradiated siblings and a survival ratio was established. At the irradiation dosage chosen, there was no apparent difference in the survival of irradiated and unirradiated control flies (survival ratio close to 1) for all irradiation times, indicating an insensitivity to this dose of irradiation. At the same dose, spnA, spnB and spnB;spnA doubly mutant embryos and larvae show an age-dependent sensitivity to ionizing radiation. When irradiated during embryogenesis (0-24 h AEL), there was little difference in survival between mutant and control. However, during later larval stages (48-72 h AEL) there was a significant difference between the survival of spnA mutant larvae and their heterozygous control siblings. This increase in sensitivity to irradiation with age is likely due to maternal proteins present in the developing embryos; as the animals get older less maternal protein is present due to degradation. Heterozygous control progeny experience the same degradation of maternal product but survive due to their ability to synthesize necessary gene product de novo. While spnA mutants show a striking sensitivity to irradiation at late third instar (48-72 h AEL), spnB mutants show only a modest sensitivity to ionizing radiation. This is in contrast to the insensitivity observed when spnB mutants were exposed to MMS (Ghabrial, 1998). spnB;spnA double mutants do not show a synergistic sensitivity to ionizing radiation, rather, they behave similar to spnA alone, suggesting that the two genes are part of the same non-redundant pathway. Most importantly, these results show that SpnA does indeed play a role in the soma to protect against chromosomal damage inflicted by DNA damaging agents (Staeva-Vieira, 2003).
To better characterize the role of SpnA during oogenesis, the meiotic defect of the spnA null allele was analyzed in detail. It was asked whether meiotic chromosome synapsis is affected in the mutants, whether DNA breaks occur during meiosis and can be repaired in the mutant, and finally whether spnA mutants indeed cause activation of a meiotic checkpoint. In Drosophila, each germline stem cell, at the anterior tip of each ovariole, divides asymmetrically to produce a new stem cell and a differentiating cystoblast. The cystoblast undergoes four rounds of mitotic division with incomplete cytokinesis to generate a cyst of 16 cells. The cells within a cyst remain interconnected by cytoplasmic bridges called ring canals. The initiation of meiosis is indicated by the appearance of the synaptonemal complex (SC), which assembles in region 2a of the germarium in the four cells of the cyst that form first and thus contain either three- or four-ring canals. The two four-ring canal cells will become the pro-oocytes as defined by the persistence of the SC and their accumulation of oocyte specific markers in region 2b. By stage 1 of oogenesis, the SC, as observed by immunostaining for the Drosophila SC component C(3)G, and oocyte markers are restricted to a single cell, the future oocyte. As the egg matures, C(3)G begins to lose association with chromatin and the SC is no longer observed (Staeva-Vieira, 2003).
The distribution of C(3)G was followed in spnA mutant germline cysts. As in the wild type, C(3)G expression is first detected in region 2A. However, C(3)G restriction to the oocyte and its dissolution from the chromatin are delayed. At stage 1 of oogenesis, while the SC is always restricted to just the oocyte in wild type, it persists from time to time in both pro-oocytes in spnA mutants, similar to what was observed previously (Huynh, 2000). Furthermore, when C(3)G staining decreases in the maturing stage 5 oocyte, it remains in the mutant. By stage 7, the staining is no longer detected in the oocyte of either wild-type or spnA mutant egg chambers. These results suggest that synapse formation is appropriately initiated in spnA mutants but the failure to repair broken DNA causes a delay in the resolution of synapsis, first in the cyst that will not become the oocyte and subsequently in the oocyte as it progresses through meiosis (Staeva-Vieira, 2003).
The meiotic phenotype of spnA suggests that there may be a delay in proper meiotic chromosome dynamics due to the failure to repair DSBs. To visualize DSBs cytologically, an antibody was used that recognizes gamma-H2AX, a phospho-epitope of the human histone H2A variant, H2AX, which becomes phosphorylated upon DSB formation. The phospho-epitope is conserved in Drosophila histone variant HIS2AV and becomes phosphorylated in the event of DSBs, whether induced exogenously or during meiosis (Madigan, 2002; Jang, 2003). During wild-type meiosis, few gamma-HIS2AV foci were observed, presumably due to the rapid repair of DSBs and the formation of viable recombination intermediates. When observed, gamma-HIS2AV foci were found in only one cell in region 2a of the germarium. This early appearance of gamma-HIS2AV, before the restriction of other oocyte markers to a single cell, suggests that the regulation of DSB formation and persistence may be a critical event in oocyte specification. gamma-HIS2AV foci were not observed from region 2B onwards. Thus, DSBs are rapidly processed and recombination intermediates are formed concomitant with oocyte specification. In contrast, spnA mutant germaria show a more robust HIS2AV activation in one or two cells of a growing cyst in region 2a, suggesting that in spnA DSBs form at the normal time but their resolution is delayed. Furthermore, gamma-HIS2AV localization is more extensive along the DNA rather than in distinct foci as observed in the wild type, possibly due to the accumulation of unresolved breaks along the chromosomes. HIS2AV activation persists in the oocyte nucleus through later stages of oogenesis suggesting a failure to properly repair DNA breaks (Staeva-Vieira, 2003).
If all the defects observed in spnA mutants are due to the activation of a checkpoint upon failure to repair DSBs, one would predict that mutations that prevent break formation in the first place would suppress the spnA phenotype. This rationale is suggested by results in yeast where mutations in spo11 suppress the meiotic sporulation defects of dmc1 mutations (Roeder, 1997; reviewed in Bishop, 1999). Subsequent to the yeast work, it was shown that the eggshell phenotype of two Drosophila Rad51 family members, spnB and spnD, as well as the Rad54 homolog, okra, is suppressed in the absence of mei-W68, the Drosophila homolog of Spo11 (Ghabrial, 1998). Therefore double mutants between mei-W68 and spnA were generated and their ability to produce properly patterned eggs was examined. Control females that were efficient at producing DSBs during meiosis [mei-W68/+; spnA093A/Df(3R)X3F] but were defective in SpnA function, produced progeny with spindle eggshells. In contrast, in flies defective in DSB production and SpnA function [mei-W68/mei-W68; spnA093A/Df(3R)X3F], the spindle phenotype was rarely observed (<1%). Furthermore, the oocyte nuclear morphology and Gurken protein localization and distribution appeared normal in the double mutants. mei-W68 also suppresses the embryonic lethality associated with loss of maternal SpnA. In this situation, embryos from doubly mutant females survived to adulthood with a frequency similar to that observed in mei-W68 progeny alone. The fact that all phenotypes associated with spnA mutants are suppressed by mei-W68 suggests that it is indeed the role of SpnA in repair of meiotic-induced DSBs that is essential for normal oogenesis and survival (Staeva-Vieira, 2003).
The data show that DSBs are readily detectable by gamma-HIS2AV staining and persist during oogenesis in spnA mutants. Unrepaired DSBs or unresolved recombination intermediates lead to the activation of an ATM/ATR-dependent cell cycle checkpoint in mitosis and meiosis, which often causes delays in cell cycle progression in order to repair DNA damage (Roeder, 2000). To test whether unrepaired DSBs or unresolved recombination intermediates in spnA mutants trigger a cell cycle checkpoint that leads to defects in oocyte development, it was desirable to inactivate the checkpoint response (Staeva-Vieira, 2003).
Two genes have been implicated in checkpoint function, the Drosophila ATR homolog Mei-41 and the Chk2 homolog DmChk2/Mnk/Loki. Since Drosophila mei-41 mutants also show a defect in meiotic recombination, it is difficult to assess the exact step in meiosis that is affected in this mutant. Focus was therefore placed on the checkpoint protein, DmChk2. On its own, chk2 mutants do not appear to have a meiotic phenotype. Females doubly mutant for chk2 and spnA produced progeny with wild-type egg shape (100 versus 6%), even a reduction in the copy number of chk2 (chk2/+, spnA057/spnA093A) partially suppresses the spindle phenotype (22 versus 94%). In addition, the karyosome appears normal in the oocytes from females doubly mutant for chk2 and spnA, suggesting that the abnormal nuclear morphology observed in spnA mutant oocytes is not the result of fragmented DNA, since the DNA breaks should persist in these double mutants. In contrast to the mei-W68;spnA doubles, deletion of chk2 did not suppress the maternal-effect embryonic lethality of spnA. Thus, it is concluded that the spnA phenotype results from the activation of a Chk2-dependent meiotic checkpoint (Staeva-Vieira, 2003).
Rad51 is a highly conserved protein throughout the eukaryotic kingdom and an essential enzyme in DNA repair and recombination. It possesses DNA binding activity and ATPase activity, and interacts with meiotic chromosomes during prophase I of meiosis. Drosophila Rad51, Spindle-A (SpnA) protein has been shown to be involved in repair of DNA damage in somatic cells and meiotic recombination in female germ cells. In this study, DNA binding activity of SpnA is demonstrated by both agarose gel mobility shift assay and restriction enzyme protection assay. SpnA is also shown to interact with meiotic chromosomes during prophase I in the primary spermatocytes of hsp26-spnA transgenic flies. In addition, SpnA is highly expressed in embryos, and the depletion of SpnA by RNA interference (RNAi) leads to embryonic lethality implying that SpnA is involved in early embryonic development. Therefore, these results suggest that Drosophila SpnA protein possesses properties similar to mammalian Rad51 homologs (Yoo, 2006).
Rad51 protein has been demonstrated to possess DNA binding activity, ATPase activity, and strand transfer activity. These properties are essential to homologous recombination since the primary function of Rad51 protein is to bring two homologous DNA molecules into close proximity to facilitate the formation of heteroduplex DNA and to mediate strand exchange between them. Thus, the presumptive first step of homologous recombination is binding of Rad51 protein to the DNA molecule. In the present study, the DNA binding activity of SpnA protein is demonstrated in a Mg2+-dependent but ATP-independent manner. A substantial body of biochemical evidence indicates that the binding reaction of Rad51 protein is affected by nucleotide cofactors, Mg2+ concentration, and salt concentration. In contrast to Mg2+ which is an indispensable factor for DNA binding assay, the precise role of ATP is still controversial. It has been reported that yeast Rad51 protein binds to both ds- and ss-DNA only in the presence of ATP and that neither ADP nor the nonhydrolyzable ATP analogues can substitute the function of ATP. In contrast, the binding of yeast Rad51 protein to DNA in the absence of nucleotide cofactor has been demonstrated at low pH condition. A study using human Rad51 protein also represented that both ds- and ss-DNA binding activities are not dependent upon a nucleotide cofactor. Therefore, it is not surprising that ATP is not an essential factor for DNA binding of SpnA protein. Recently, the Drosophila Rad51 homolog, SpnA protein is also shown to be involved in the strand exchange step. Although the precise biochemical properties of SpnA protein remain to be determined, these results strongly support the central role of SpnA in repair of DSBs by homologous recombination (Yoo, 2006).
Several studies using mouse spermatocytes and oocytes have reported that Rad51 foci appear as early as premeiotic S phase before the initiation of synapsis. These foci are increased in number and become organized during leptotene, and then, dramatically decreased as pachytene progresses. In this study, SpnA foci were also observed during early prophase I and rapidly disappeared at late prophase I of the spermatocytes of hsp26-spnA transformants. Although it is not a physiological condition, the time course of appearance of SpnA and its distribution in spermatocytes are similar to mammalian Rad51 homologs. The failure to detect SpnA foci in wild type spermatocytes suggest that SpnA might be absent or, if any, at very low level in the spermatocytes. A previous study also reported no role of SpnA in male meiotic chromosome pairing, a diagnostic phenotype in meiotic recombination. Taken together with the observation that spnA null mutant males are fertile, SpnA may be dispensable during Drosophila spermatogenesis unlike mammalian Rad51 proteins (Yoo, 2006).
The disruption of rad51 gene in mice results in early embryonic lethality, indicating a crucial role of Rad51 in embryogenesis. Since the cells are rapidly dividing during embryogenesis, Rad51 might be required for the cell proliferation, cell cycle control, DNA replication, or transcription possibly by interacting with p53, Brca1, or Brca2. However, the precise role of Rad51 protein during embryogenesis is not understood yet. In contrast to mammals, Drosophila spnA null mutants are viable although they are defective in recombination and DNA repair. The survival of Drosophila mutants might be explained by the presence of maternal SpnA proteins for early embryonic development. The depletion of maternally stored spnA transcript may demonstrate a possible role of SpnA in rapidly dividing cells during early development. Double mutants for spnA and mei-W68 escape the complete sterility of spnA mutant females caused by defects in DNA repair during oogenesis. mei-W68 is the Drosophila homolog of Spo11 that induces the DSB formation. Since spnA phenotypes are suppressed by mei-W68 mutation, the double mutants are fertile and their progeny survive to adulthood, suggesting that SpnA may not be essential for viability. In this study, in an attempt to examine the phenotype caused by the depletion of SpnA solely, the maternal effect of SpnA was eliminated by employing RNAi technique. Interestingly, the results showed that both dsRNA and siRNA targeting spnA severely interfered with normal development, implying a role of SpnA during embryogenesis. It is possible that SpnA functions in the repair of mis-incorporated nucleotides during DNA replication or in the removal of nucleotide wastes during embryogenesis. Although many SpnA foci associate with unknown material stained with DAPI during mitosis, the role of SpnA in embryonic development is unclear and need to be addressed in the future experiments (Yoo, 2006).
When exposed to DNA-damaging agents, components of the DNA damage response (DDR) pathway trigger apoptosis, cell cycle arrest and DNA repair. Although failures in this pathway are associated with cancer development, the tumor suppressor roles of cell cycle arrest and apoptosis have recently been questioned in mouse models. Using Drosophila epithelial cells that are unable to activate the apoptotic program, evidence is provided that ionizing radiation (IR)-induced DNA damage elicits a tumorigenic behavior in terms of E-cadherin delocalization, cell delamination, basement membrane degradation and neoplasic overgrowth. The tumorigenic response of the tissue to IR is enhanced by depletion of Okra/DmRAD54 or spnA/DmRAD51-genes required for homologous recombination (HR) repair of DNA double-strand breaks in G2-and it is independent of the activity of Lig4, a ligase required for nonhomologous end-joining repair in G1. Remarkably, depletion of Grapes/DmChk1 or Mei-41/dATR-genes affecting DNA damage-induces cell cycle arrest in G2-compromises DNA repair and enhances the tumorigenic response of the tissue to IR. On the contrary, DDR-independent lengthening of G2 has a positive impact on the dynamics of DNA repair and suppressed the tumorigenic response of the tissue to IR. These results support a tumor suppressor roles of apoptosis, DNA repair by HR and cell cycle arrest in G2 in simple epithelia subject to IR-induced DNA damage (Dekanty, 2014).
A first year graduate student, Ann Dee Margulies, changed my research career in 1962 by challenging me to direct her in the isolation of recombination-deficient mutants of Escherichia coli K-12. She succeeded in isolating two mutants, which conjugated with donor strains and received the donor DNA, but could not recombine that DNA with their own chromosomes. Ann Dee showed that both mutants were much more sensitive to UV radiation than was the wild type. Furthermore, she showed that one of these mutants carried a single mutation affecting both recombination and resistance. This work, published in 1965, was the first demonstration of the recA gene of E. coli. Subsequent work led to the discovery of many more recombination genes, the phenomenon of post replication-recombination repair, the invention of the SOS hypothesis and the discovery of genes encoding proteins with similar primary structure and function in all major groups of organisms (Clark, 1996).
Germline specific expression and the sterility of DMC1-deficient mice defined DMC1 as a meiosis-specific component of the homologous recombination complex (Habu, 1996; Pittman, 1998). However, genome-wide search failed to identify a clear Dmc1 homolog in Drosophila. Considering that a Dmc1-like gene would be expressed exclusively in the germline, whether DmRad51/SpnA or any of the other Drosophila Rad51 family members are meiosis specific was examined by determining germline and soma gene expression of the five Drosophila Rad51 genes by RT-PCR. Rad51 gene expression profiles from wild-type females, flies from tudor mutant females, which lack germline, and to males, which fail to undergo meiotic recombination, were compared. For each Rad51 gene, primers were designed to specifically amplify a fragment of the corresponding transcript. As a control for germline-specific expression, oskar and nanos RNA were examined by RT-PCR. spnA, spnB and rad51C RNA are expressed in both males and females. Furthermore, their expression is not limited to the female germline. Interestingly, rad51D and spnD RNA appear to be expressed almost exclusively in the germline of adult females. Thus, Drosophila may have two Rad51 family members that are specifically involved in meiotic recombination and functionally equivalent to Dmc1 (Staeva-Vieira, 2003).
To clarify RAD51 interactions controlling homologous recombination, the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus is reported. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P. furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination (Shin, 2003).
The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. The rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, Rad51 protein is found to be similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae (Shinohara, 1992).
The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. The Rad54 protein interacts with the Rad51 protein both in vivo and in vitro, and the NH2-terminal 115 residues of the Rad54 protein are shown to be necessary for this interaction. These data imply that the Rad54 protein is part of a multiprotein yeast recombination complex (Jiang, 1996).
Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases. Both proteins are also important members of the RAD52 group, which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae. Rad51 and Rad54 proteins are shown by genetic, molecular and biochemical criteria to interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression is observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. These data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. Since both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms (Clever, 1997).
The Schizosaccharomyces pombe rhp51+, rad22+ and rhp54+ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Delta and rhp54Delta strains are extremely sensitive to ionizing radiation; the rad22Delta mutant turns out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Delta and rhp54Delta are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Delta mutant is only reduced by a factor of two. Blot-analysis demonstrates that the majority of the leu+ transformants of the wild-type and rad22Delta strains have integrated one or more copies of the vector. Gene conversion events are observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Delta and a rhp54Delta background. Meiotic recombination is hardly affected in the rad22Delta mutant. The rhp51Delta and rhp54Delta strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Delta strain and 27% in the rhp54Delta strain, as compared with wild-type cells. However, in the rhp51Delta mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe (Muris, 1997).
The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. Rad52 protein is found to stimulate DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required (New, 1998).
The Saccharomyces cerevisiae RAD51 and RAD54 genes are both required for the occurrence of homologous recombination and for the repair of double-stranded DNA breaks. Previous studies have indicated that Rad51 protein, together with the single-stranded DNA-binding factor replication protein A (RPA), can promote the formation of heteroduplex DNA, which is a key intermediate in homologous recombination. Rad54 protein has now been purified to near homogeneity and its molecular function has been biochemically tested. Rad54 protein possesses a double-stranded DNA-dependent ATPase activity, and it interacts with the Rad51 protein. Addition of Rad54 protein to reactions containing Rad51 strongly stimulates the rate of pairing between homologous single-stranded and double-stranded DNA molecules. It is concluded that Rad54 acts to overcome kinetic impediments that would limit homologous DNA pairing between recombining chromosomes in vivo (Petukhova, 1998).
Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA (Petukhova, 1999).
In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homolog of the recA gene; the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologs of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Rad52 protein is shown to stimulate Rad51 reactions and binding to Rad51 is necessary for this stimulatory effect. It is concluded that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments (Shinohara, 1998).
Yeast Rad51 recombinase has only minimal ability to form D loop. Addition of Rad54 renders D loop formation by Rad51 efficient, even when topologically relaxed DNA is used as substrate. Treatment of the nucleoprotein complex of Rad54 and relaxed DNA with topoisomerases reveals dynamic DNA remodeling to generate unconstrained negative and positive supercoils. DNA remodeling requires ATP hydrolysis by Rad54 and is stimulated by Rad51-DNA nucleoprotein complex. A marked sensitivity of DNA undergoing remodeling to P1 nuclease indicates that the negative supercoils produced lead to transient DNA strand separation. Thus, a specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation (Van Komen, 2000).
RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carries out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibits dsDNA-specific ATPase activity. Rad54 protein stimulates Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation is accompanied by a concurrent increase in the formation of heteroduplex DNA. These results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein does not stimulate DNA strand exchange by increasing presynaptic complex formation. It is concluded that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments (Solinger, 2001a).
Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depends on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins (Solinger, 2001b).
Yeast Rad51 promotes homologous pairing and strand exchange in vitro, but this activity is inefficient in the absence of the accessory proteins, RPA, Rad52, Rad54 and the Rad55-Rad57 heterodimer. A class of rad51 alleles was isolated that suppresses the requirement for RAD55 and RAD57 in DNA repair, but not the other accessory factors. Five of the six mutations isolated map to the region of Rad51 that by modeling with RecA corresponds to one of the DNA-binding sites. The other mutation is in the N-terminus of Rad51 in a domain implicated in protein-protein interactions and DNA binding. The Rad51-I345T mutant protein shows increased binding to single- and double-stranded DNA, and is proficient in displacement of replication protein A (RPA) from single-stranded DNA, suggesting that the normal function of Rad55-Rad57 is promotion and stabilization of Rad51-ssDNA complexes (Fortin, 2002).
Rad54 protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. This study addresses the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. Saccharomyces cerevisiae Rad54 is shown to exert distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 protein but to a lesser extent. In contrast, the interaction of Rad54 protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 protein hydrolyzes ATP in a reduced mode at 60%-80% of its rate on protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. It is suggested that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination (Kiianitsa, 2002).
Saccharomyces cerevisiae Rad51, Rad54, and replication protein A (RPA) proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating single-stranded DNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities. RPA is still indispensable for optimal reaction efficiency, but its role in this instance is to sequester free single-stranded DNA, which otherwise inhibits Rad51 and Rad54 functions. Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction (Van Komen, 2002).
Repairing a double-strand break by homologous recombination requires binding of the strand exchange protein Rad51p to ssDNA, followed by synapsis with a homologous donor. Chromatin immunoprecipitation has been used to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HMLα donor. Localization of Rad51p to MAT precedes its association with HML, providing evidence of the time needed for the Rad51 filament to search the genome for a homologous sequence. Rad51p binding to ssDNA requires Rad52p. The absence of Rad55p delays Rad51p binding to ssDNA and prevents strand invasion and localization of Rad51p to HMLα. Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HMLα; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand (Sugawara, 2003).
Repair of a double-strand break (DSB) often occurs by gene conversion, a homologous recombination event. Homologous recombination depends on a search for homology by the ends of the broken chromosome to locate an intact donor sequence that could be used as a template for DNA repair. In budding yeast and other eukaryotes, the search for homology is facilitated by the DNA strand exchange protein, Rad51p, the homolog of the bacterial RecA protein. In vitro, Rad51p, like RecA, forms extensive Rad51p-DNA filaments, with 3 bp bound per monomer. The RecA or Rad51p filament can catalyze strand exchange between ssDNA and a homologous dsDNA. This process is greatly facilitated by prior exposure of the ssDNA to a ssDNA binding protein, SSB in bacteria, or RPA in eukaryotes. These ssDNA binding proteins appear to facilitate the polymerization of RecA/Rad51p across ssDNA regions that can form secondary structure (Sugawara, 2003 and references therein).
Incubation of RPA with ssDNA before the addition of Rad51p, however, reduces formation of Rad51 filaments. Either Rad52p or the Rad55p/Rad57p heterodimer can overcome this inhibition and mediate the loading of Rad51p onto ssDNA. Rad54p shares sequence similarity with the Swi2/Snf2 family of chromatin-remodeling proteins and appears to act at a later step in strand exchange, to extend heteroduplex DNA, and to alter DNA conformation during the synaptic and/or postsynaptic phases of strand exchange. In vivo, Rad51p-mediated recombination in S. cerevisiae requires the participation of Rad52p, Rad54p, Rad55p, and Rad57p. Deletions of RAD52 are the most defective in spontaneous and DSB-induced recombination, since this protein is required both for Rad51p-mediated and Rad59p-mediated recombination events. The requirements for Rad55p and Rad57p are the least stringent, as defects are often seen only at low temperature. This defect can be suppressed by the overexpression of Rad51p (Sugawara, 2003 and references therein).
One of the best-studied homologous recombination events is the HO endonuclease-induced switching of the MATa locus, using HMLα as the donor template during gene conversion. A galactose-inducible GAL::HO gene provides the means to induce the DSB synchronously in all cells of the population. Physical monitoring of MAT DNA has shown that the DSB ends are first resected by 5' to 3' exonucleases, presumably so that the 3'-ended ssDNA can recruit the Rad51p strand exchange protein and undergo strand invasion of the donor and initiate new DNA synthesis. Although the formation of a strand invasion intermediate has not been directly demonstrated, the next step in the process, the use of the 3' end of the invading strand as a primer to copy the template DNA, has been detected by PCR. This was observed to occur about 30 min after the appearance of the DSB and 30 min before the recombination event was completed. HO cleavage and 5' to 3' resection are normal in mutant cells lacking the auxiliary recombination proteins Rad52p, Rad54p, Rad55p, and Rad57p, but the primer extension step does not occur; however, in some strain backgrounds there is a small amount of MAT switching in rad54Δ mutants (Sugawara, 2003 and references therein).
To determine more precisely the recombination steps that are prevented in the absence of the auxiliary proteins, chromatin immunoprecipitation (ChIP) was used to follow the ability of Rad51p to associate with ssDNA formed at MAT and with DNA sequences at HML as well as a model for the synapsis of MAT and HML during gene conversion. This approach has enabled distinct functions for Rad52p, Rad54p, and Radd55p/Rad57p to be seen in the Rad51p-mediated process. It is concluded that Rad52p is required for the recruitment of Rad51p to a DSB, whereas the rate of recruitment is reduced or delayed in the absence of Rad55p/Rad57p. At the step of strand exchange, Rad55p/Rad57p is required for recruitment of Rad51p to the donor whereas Rad54p is not, suggesting that synaptic association between MAT and the HML donor does not need Rad54p. Rad54p is required at a postsynaptic step to enable the completion of DNA repair (Sugawara, 2003).
In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, Rad52, Rad55 and Rad57 proteins. In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein. Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dependent ATPase activity and changes DNA topology in an ATP hydrolysis-dependent manner. Rad54 catalyzes bidirectional nucleosome redistribution by sliding nucleosomes along DNA. Nucleosome redistribution is greatly stimulated by the Rad51 nucleoprotein filament but does not require the presence of homologous single-stranded DNA within the filament. On the basis of these data, it is proposed that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination (Alexeev, 2003).
Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. It is suggested that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p (Lee, 2003).
To prevent genome instability, recombination between sequences that contain mismatches (homeologous recombination) is suppressed by the mismatch repair (MMR) pathway. To understand the interactions necessary for this regulation, the genetic requirements for the inhibition of homeologous recombination were examined using mutants in the RAD52 epistasis group of Saccharomyces cerevisiae. The use of a chromosomal inverted-repeat recombination assay to measure spontaneous recombination between 91% and 100% identical sequences demonstrates differences in the fidelity of recombination in pathways defined by their dependence on RAD51 and RAD59. In addition, the regulation of homeologous recombination in rad51 and rad59 mutants displays distinct patterns of inhibition by different members of the MMR pathway. Whereas the requirements for the MutS homolog, MSH2, and the MutL homolog, MLH1, in the suppression of homeologous recombination are similar in rad51 strains, the loss of MSH2 causes a greater loss in homeologous recombination suppression than does the loss of MLH1 in a rad59 strain. The nonequivalence of the regulatory patterns in the wild-type and mutant strains suggests an overlap between the roles of the RAD51 and RAD59 gene products in potential cooperative recombination mechanisms used in wild-type cells (Spell, 2003).
A chromosome fragmentation assay was used to measure the efficiency and genetic control of break-induced replication (BIR) in Saccharomyces cerevisiae. Formation of a chromosome fragment by de novo telomere generation at one end of the linear vector and recombination-dependent replication of 100 kb of chromosomal sequences at the other end of the vector occurred at high frequency in wild-type strains. RAD51 was required for more than 95% of BIR events involving a single-end invasion and was essential when two BIR events were required for generation of a chromosome fragment. The similar genetic requirements for BIR and gene conversion suggest a common strand invasion intermediate in these two recombinational repair processes. Mutation of RAD50 or RAD59 conferred no significant defect in BIR in either RAD51 or rad51 strains. RAD52 is essential for BIR at unique chromosomal sequences, although rare recombination events were detected between the subtelomeric Y' repeats (Davis, 2004).
The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic interhomologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. This study describes an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, it has been demonstrated that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. Evidence is presented that the Rad51 binding domain resides within the N terminus of Rdh54. Accordingly, N-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54Delta mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54.Rad51 complex formation. The Rdh54 expression and purification procedures described in this study should facilitate the functional dissection of this DNA recombination/repair factor (Chi, 2006).
Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Evidence is provided that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, a class of rad52 mutants is described that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of these conclusions, it is shown that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells (Lettier, 2006; full text of article).
Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, in vivo functions were examined of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Δ mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Δ mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Δ and rhp57Δ mutants and completely abolished in an swi5Δ rhp57Δ double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator (Akamatsu, 2007).
Meiotic recombination-related DNA synthesis (MRDS) was analyzed in Saccharomyces cerevisiae by specifically timed incorporation of thymidine analogs into chromosomes. Lengths and positions of incorporation tracts were determined relative to a known recombination hot spot along DNA, as was the timing and localization of incorporation relative to forming and formed synaptonemal complex in spread chromosomes. Distinct patterns could be specifically associated with the majority cross-over and non-cross-over recombination processes. The results obtained provide direct evidence for key aspects of current consensus recombination models, provide information regarding temporal and spatial relationships between non-cross-over formation and the synaptonemal complex, and raise the possibility that removal of RecA homolog Rad51 plays a key role in regulating onset of MRDS. Finally, classical observations on MRDS in Drosophila, mouse, and lily are readily mapped onto the findings presented in this study, providing further evidence for a broadly conserved meiotic recombination process (Terasawa, 2007).
Heterochromatin plays a key role in protection of chromosome integrity by suppressing homologous recombination. In Saccharomyces cerevisiae, Sir2p, Sir3p, and Sir4p are structural components of heterochromatin found at telomeres and the silent mating-type loci. This study investigated whether incorporation of Sir proteins into minichromosomes regulates early steps of recombinational repair in vitro. It was found that addition of Sir3p to a nucleosomal substrate is sufficient to eliminate yRad51p-catalyzed formation of joints, and that this repression is enhanced by Sir2p/Sir4p. Importantly, Sir-mediated repression requires histone residues that are critical for silencing in vivo. Moreover, the SWI/SNF chromatin-remodeling enzyme facilitates joint formation by evicting Sir3p, thereby promoting subsequent Rad54p-dependent formation of a strand invasion product. These results suggest that recombinational repair in the context of heterochromatin presents additional constraints that can be overcome by ATP-dependent chromatin-remodeling enzymes (Sinha, 2009).
Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homolog the Escherichia coli RecA protein, the DNA binding properties of the Rad51 protein were analyzed. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of protein. Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6-9 nucleotides or base pairs. Therefore, Rad51 protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, it is concluded that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it (Zaitseva, 1999).
Crossover and noncrossover recombinants can form by two different pathways during meiotic recombination in Saccharomyces cerevisiae. The MER3 gene is known to affect selectively crossover, but not noncrossover, recombination. The Mer3 protein is a DNA helicase that unwinds duplex DNA in the 3' to 5' direction. To define the underlying molecular steps of meiotic recombination, the role of Mer3 helicase in DNA strand exchange promoted by Rad51 protein was investigated. It was found that Mer3 helicase does not function as an initiator of DNA pairing events but, rather, it stimulates DNA heteroduplex extension in the 3' -> 5' direction, relative to the incoming (or displaced) single-stranded DNA. Conversely, Mer3 helicase blocks DNA heteroduplex extension in the 5' -> 3' direction. These results support the idea that Mer3 helicase stabilizes nascent joint molecules via DNA heteroduplex extension to permit capture of the second processed end of a double-stranded DNA break, a step that is required for crossover recombinant product formation (Mazina, 2004).
The meiosis-specific recombinase Dmc1 plays a critical role in DNA strand exchange in budding yeast. Tid1/Rdh54, a member of the Swi2/Snf2 family of DNA translocases, has been shown to stimulate Dmc1-dependent recombination. Tid1 and its budding yeast paralog Rad54 have a variety of biochemical activities that may contribute to their biological function. This study demonstrates that Dmc1 can associate with chromatin in the absence of DNA double-strand breaks (DSBs), and Tid1 suppresses this association. Chromatin immunoprecipitation experiments indicate that an activity shared by Tid1 and Rad54 is required for normal assembly of Dmc1 at DSB sites in preparation for recombination. These results lead to a model in which the ATP hydrolysis-dependent DNA translocase activity of Tid1 acts to promote dissociation of Dmc1 from nonreombinogenic sites on chromatin, with Rad54 being able to substitute for this function in the absence of Tid1. The tendency of Dmc1 to form unproductive interactions with chromatin is proposed to be a consequence of the mechanism of strand exchange. The results raise the possibility that ATP hydrolysis-dependent disruption of nonproductive recombinase-DNA interactions is a feature shared with other homologous recombination systems (Holzen 2006).
DMC1 is a new meiosis-specific yeast gene. Dmc1 protein is structurally similar to bacterial RecA proteins. dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase. dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related. dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation. dmc1 mutations confer prophase arrest. Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle 'checkpoint' in response to a primary defect in prophase chromosome metabolism. DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism (Bishop, 1992).
DMC1, the meiosis-specific eukaryotic homolog of bacterial recA, is required for completion of meiotic recombination and cell cycle progression past prophase. In a dmc1 mutant, double strand break recombination intermediates accumulate and cells arrest in prophase. Genes have been isolated which, when present at high copy numbers, suppress the meiotic arrest phenotype conferred by dmc1 mutations. Among the genes isolated were two which suppress arrest by altering the recombination process. REC114 suppresses formation of double strand break (DSB) recombination intermediates. The low viability of spores produced by dmc1 mutants carrying high copy numbers of REC114 is rescued when reductional segregation is bypassed by mutation of spo13. High copy numbers of RAD54 suppress dmc1 arrest, promote DSB repair, and allow formation of viable spores following reductional segregation. Analysis of the combined effects of a null mutation in RED1, a gene required for meiotic chromosome structure, with null mutations in RAD54 and DMC1 shows that RAD54, while not normally important for repair of DSBs during meiosis, is required for efficient repair of breaks by the intersister recombination pathway that operates in red1 dmc1 double mutants. It is concluded that over-expression of REC114 suppresses meiotic arrest by preventing formation of DSBs. High copy numbers of RAD54 activate a DMC1-independent mechanism that promotes repair of DSBs by homology-mediated recombination. The ability of RAD54 to promote DMC1-independent recombination is proposed to involve suppression of a constraint that normally promotes recombination between homologous chromatids rather than sisters (Bishop, 1999).
Dmc1 and Rad51 are eukaryotic RecA homologs that are involved in meiotic recombination. The expression of Dmc1 is limited to meiosis, whereas Rad51 is expressed in mitosis and meiosis. Dmc1 and Rad51 have unique and overlapping functions during meiotic recombination. Dmc1 protein has been purified from the budding yeast Saccharomyces cerevisiae and its biochemical activity has been characterized. The protein has a weak DNA-dependent ATPase activity and binds both single-strand DNA (ssDNA) and double-strand DNA. Electrophoretic mobility shift assays suggest that DNA binding by Dmc1 is cooperative. Dmc1 renatures linearized plasmid DNA with first order reaction kinetics and without requiring added nucleotide cofactor. In addition, Dmc1 catalyzes strand assimilation of ssDNA oligonucleotides into homologous supercoiled duplex DNA in a reaction promoted by ATP or the non-hydrolyzable ATP analogue AMP-PNP (Hong, 2001).
Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. Budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, targeting of Mph1 to double-strand breaks was found in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from this analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks (Prakash, 2009).
Since Mph1 influences the outcome rather than the efficiency of recombinational repair events, it very likely acts by shunting a DNA intermediate into the non-crossover generating synthesis-dependent strand annealing pathway. As revealed in ChIP experiments, Mph1 is targeted to DSBs in cells, suggesting that its action in recombination regulation is direct. Biochemical results provided evidence that Mph1 regulates recombination pathway choice by dissociating the invading DNA strand from the Rad51-made D-loop (Prakash, 2009).
XRCC3 is a RAD51 paralog that functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). XRCC3 mutation causes severe chromosome instability. XRCC3 mutant cells display radically altered HR product spectra, with increased gene conversion tract lengths, increased frequencies of discontinuous tracts, and frequent local rearrangements associated with HR. These results indicate that XRCC3 function is not limited to HR initiation, but extends to later stages in formation and resolution of HR intermediates, possibly by stabilizing heteroduplex DNA. The results further demonstrate that HR defects can promote genomic instability not only through failure to initiate HR (leading to nonhomologous repair) but also through aberrant processing of HR intermediates. Both mechanisms may contribute to carcinogenesis in HR-deficient cells (Brennaman, 2002).
Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, mammalian expression vectors were generated that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to those in wild-type-complemented clones. Because of the mutants dysfunction, it is proposed that ATP binding and hydrolyzing activities of XRCC3 are essential. In vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C was tested upon co-expression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in homologous recombination repair (Yamada, 2004).
The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive Rad51d-/- Trp53-/- primary mouse embryonic fibroblasts (MEFs) exhibit telomeric DNA repeat shortening compared to Trp53-/- or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also results in telomere erosion and chromosome fusion. It is concluded that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion (Tarsounas, 2004).
Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. This study clarifies the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. These findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing (Krejci, 2003).
Homologous recombination is a ubiquitous process with key functions in meiotic and vegetative cells for the repair of DNA breaks. It is initiated by the formation of single-stranded DNA on which recombination proteins bind to form a nucleoprotein filament that is active in searching for homology, in the formation of joint molecules and in the exchange of DNA strands. This process contributes to genome stability but it is also potentially dangerous to cells if intermediates are formed that cannot be processed normally and thus are toxic or generate genomic rearrangements. Cells must therefore have developed strategies to survey recombination and to prevent the occurrence of such deleterious events. In Saccharomyces cerevisiae, genetic data have shown that the Srs2 helicase negatively modulates recombination, and later experiments suggested that it reverses intermediate recombination structures. DNA strand exchange mediated in vitro by Rad51 is inhibited by Srs2, and Srs2 disrupts Rad51 filaments formed on single-stranded DNA. These data provide an explanation for the anti-recombinogenic role of Srs2 in vivo and highlight a previously unknown mechanism for recombination control (Veaute, 2003).
S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by preventing unscheduled mitotic recombination events. The RecQ helicase-mediated mechanisms controlling genome stability by analyzing replication forks encountering a damaged template were examined in sgs1 cells. In sgs1 mutants, recombination-dependent cruciform structures accumulate at damaged forks. Their accumulation requires Rad51 protein; this is counteracted by Srs2 DNA helicase, and does not prevent fork movement. Sgs1, but not Srs2, promotes resolution of these recombination intermediates. A functional Rad53 checkpoint kinase that is known to protect the integrity of the sister chromatid junctions is required for the accumulation of recombination intermediates in sgs1 mutants. Finally, top3 and top3 sgs1 mutants accumulate the same structures as sgs1 cells. It is suggested that, in sgs1 cells, the unscheduled accumulation of Rad51-dependent cruciform structures at damaged forks result from defective maturation of recombination-dependent intermediates that originate from the replication-related sister chromatid junctions. These findings might contribute to explaining some of the recombination defects of BLM cells (Liberi, 2005).
In this study, cell cycle-dependent expression of human and rodent RAD51 and RAD52 proteins was monitored using two approaches. (1) Flow cytometric measurements of DNA content and immunofluorescence were used to determine the phase-specific levels of RAD51 and RAD52 protein expression in irradiated and control populations. The expression of both proteins is lowest in G0/G1, increases in S and reaches a maximum in G2/M. No difference is found in the whole-cell level of RAD51 or RAD52 protein expression between gamma-irradiated and control cell populations. (2) Cell cycle-dependent protein expression was confirmed by Western analysis of populations synchronized in G0, G1 and G2 phases. Analysis of V3, a hamster equivalent of SCID, indicates that the protein level increases of RAD51 and RAD52 from G0 to G1/S/G2 do not require DNA-PK (Chen, 1997).
Rad51, a eukaryotic RecA homolog, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 foci, typically during the S phase. However, the topological relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. This study reports the results of ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. The preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin is demonstrated using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin (Tashiro, 2000).
The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome (Trojanek, 2003).
The human testis Rad51 protein, a structural homolog of E. coli RecA, binds single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using circular ssDNA and linear dsDNA (3.0 kb in length), it has been demonstrated that hRad51 promotes homologous pairing and strand exchange reactions in vitro. Joint molecule formation is dependent upon ATP hydrolysis and DNA homology and is stimulated by the single-strand DNA-binding protein RP-A. In these reactions, the 5' terminus of the complementary strand of the linear duplex is efficiently transferred to the ssDNA. However, under standard conditions, extensive strand exchange is not observed. These results establish hRad51 as a functional homolog of RecA, but indicate that the human protein and its bacterial counterpart differ in their ability to promote extensive strand transfer. It is proposed that hRad51 mediates homology recognition and initiates strand exchange, but that extensive heteroduplex formation in higher organisms requires the actions of additional proteins (Baumann, 1996).
Human homologs of RAD50, RAD51, RAD52, RAD54 and MRE11 have been identified. Targeted disruption of the murine RAD51 gene results in an embryonic lethal phenotype, indicating that Rad51 protein is required during cell proliferation. Biochemical studies have shown that human RAD51 encodes a protein of relative molecular mass 36,966 (hRad51) that promotes ATP-dependent homologous pairing and DNA strand exchange. As a structural and functional homolog of the RecA protein from Escherichia coli, hRad51 is thought to play a central role in recombination. Yeast Rad51 has been shown to interact with Rad52 protein, as does the human homolog. hRad52 is shown to stimulate homologous pairing by hRad51. The DNA-binding properties of hRad52 indicate that Rad52 is involved in an early stage of Rad51-mediated recombination (Benson, 1998).
Yeast rad51 mutants are viable, but extremely sensitive to gamma-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homolog is lethal, indicating an essential role of Rad51 in vertebrate cells. Clones of the chicken B lymphocyte line DT40 were generated carrying a human RAD51 transgene under the control of a repressible promoter and subsequently the endogenous RAD51 loci were disrupted. Upon inhibition of the RAD51 transgene, Rad51- cells accumulate in the G2/M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51- cells carry isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes (Sonada, 1998).
The repair of potentially lethal DNA double-stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3'-single-stranded DNA (ssDNA) tail. Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3'-end of the ssDNA, a characteristic that the bacterial pairing protein RecA possesses. Unexpectedly, it has been found that for the eukaryotic homolog, Rad51 protein, the 5'-end of ssDNA is more invasive than the 3'-end. This pairing bias is unaffected by Rad52, Rad54 or Rad55-57 proteins. However, further investigation reveals that, in contrast to RecA protein, the preferred DNA substrate for Rad51 protein is not ssDNA but rather dsDNA with ssDNA tails. This important distinction permits the Rad51 proteins to promote DNA strand invasion using either 3'- or 5'-ends with similar efficiency (Mazin, 2000a).
Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. It is concluded that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process (Mazin, 2000b).
Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. In this study, using physical criteria, the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein has been demonstrated directly. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein (Mazin, 2003).
The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them. Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51. However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different. When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51. For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis. Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, i.e., the reaction can get stuck. Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate (Rice, 2001).
Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors (Sigurdsson, 2001).
Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homolog, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. This study investigates the mechanistic basis for this stimulation. High ionic strength favors the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity (Liu, 2004b).
Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. Nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA were analyzed by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination (Ristic, 2005; full text of article).
NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Mouse Nbs1-null embryonic fibroblasts and embryonic stem cells were constructed through the Cre-loxP and sequential gene targeting techniques. Cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways (Yang, 2006).
GFP-Rad51 fusion proteins have been visualized in the nucleus of living cells to demonstrate the dynamic compartmentalization of Rad51 by self-association or by binding to BRCA2. Mutants of Rad51 that fail to oligomerize and/or to bind BRCA2 distinguish three fractions of Rad51 within the nucleoplasm: a relatively mobile fraction, an immobile oligomerized fraction, and an immobile BRCA2-bound fraction. Strikingly, inhibition of replication by hydroxyurea reduces the immobile fraction of nucleoplasmic Rad51. This effect is specific to Rad51 mutants that retain the capacity to bind BRCA2, indicating that the BRCA2-bound fraction is selectively mobilized. It is proposed that arrested replication triggers a switch between dual functions of BRCA2 in sequestering or mobilizing a small fraction of nucleoplasmic Rad51 and suggest a mechanism for the dynamic control of protein complexes that participate in homologous recombination (Yu, 2003).
BRCA2 is a breast tumor susceptibility gene encoding a 390-kDa protein with functions in maintaining genomic stability and cell cycle progression. Evidence has been accumulated to support the concept that BRCA2 has a critical role in homologous recombination of DNA double-stranded breaks by interacting with RAD51. In addition, BRCA2 may have chromatin modifying activity through interaction with a histone acetyltransferase protein, p300/CBP-associated factor (P/CAF). To explore how the functions of BRCA2 may be regulated, the post-translational modifications of BRCA2 throughout the cell cycle were examined. It was found that BRCA2 is hyperphosphorylated specifically in M phase and becomes dephosphorylated as cells exit M phase and enter interphase. This specific phosphorylation of BRCA2 was not observed in cells treated with DNA-damaging agents. Systematic mapping of the potential mitosis specific phosphorylation sites revealed the N-terminal 284 amino acids of BRCA2 (BR-N1) as the major region of phosphorylation and mass spectrometric analysis identified two phosphopeptides that contain 'phosphorylation consensus motifs' for Polo-like kinase 1 (Plk1). Phosphorylation of BR-N1 with Plk1 recapitulates the electrophoretic mobility change as seen in BR-N1 isolated from M phase cells. Plk1 interacts with BRCA2 in vivo, and mutation of Ser193, Ser205/206, and Thr203/207 to Ala in BR-N1 abolishes Plk1 phosphorylation, suggesting that BRCA2 is the substrate of Plk1. Furthermore, both the hyperphosphorylated and hypophosphorylated forms of BRCA2 bind to RAD51, whereas the M phase hyperphosphorylated form of BRCA2 no longer associates with the P/CAF, suggesting that the dissociation of P/CAF-BRCA2 complex is regulated by phosphorylation. Taken together, these results implicate a potential role of BRCA2 in modulating M phase progression (Lin, 2003).
Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. The interrelationship was studied between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, the efficiency of repair was analyzed in mammalian cells in which double-strand break (DSB) repair components were disrupted. An inverse relationship was observed between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, it was found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, it was found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. The role of RAD52 in homologous repair was examined. In contrast to yeast, Rad52-/- mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair (Stark, 2004; full text of article).
The ubiquitin (Ub)-conjugating enzyme Ubc13 is implicated in Rad6/Rad18-dependent postreplication repair (PRR) in budding yeast, but its function in vertebrates is not known. Disruption or siRNA depletion of UBC13 in chicken DT40 or human cells confers severe growth defects due to chromosome instability, and hypersensitivity to both UV and ionizing radiation, consistent with a conserved role for Ubc13 in PRR. Remarkably, Ubc13-deficient cells are also compromised for DNA double-strand break (DSB) repair by homologous recombination (HR). Recruitment and activation of the E3 Ub ligase function of BRCA1 and the subsequent formation of the Rad51 nucleoprotein filament at DSBs are abolished in Ubc13-deficient cells. Furthermore, generation of ssDNA/RPA complexes at DSBs is severely attenuated in the absence of Ubc13. These data reveal a critical and unexpected role for vertebrate Ubc13 in the initiation of HR at the level of DSB processing (Zhao, 2007).
A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. This study reports a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. These results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. It is proposed that PTEN acts as a guardian of genome integrity (Shen, 2007).
Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. hRad54 is able to promote transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. These results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells (Sigurdsson, 2002).
In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, these results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin (Jaskelioff, 2003).
These results suggest that Rad54p is an extremely versatile recombination protein that plays key roles in several steps of homologous recombination. Rad54p is required for optimal recruitment of Rad51p to a double strand break in vivo, and likewise Rad54p can promote formation of the presynaptic filament in vitro by helping Rad51p contend with the inhibitory effects of the ssDNA-binding protein replication protein A.2. Several studies over the past few years have also shown that the ATPase activity of Rad54p plays key roles subsequent to formation of the presynaptic filament. For instance, Rad54p is required for the Rad51p-nucleoprotein filament to form a heteroduplex joint DNA molecule, even when the homologous donor is naked DNA. In this case, it has been proposed that Rad54p might use the free energy from ATP hydrolysis to translocate along DNA, which facilitates the homology search process. This DNA-translocation model is fully consistent with findings that Rad54p can displace a DNA triplex and that the ATPase activity of Rad54p is proportional to DNA length. Rad54p also stimulates heteroduplex DNA extension of established joint molecules. Finally, Rad54p is required for Rad51p-dependent heteroduplex joint molecule formation with a chromatin donor. In this case, the results suggest that the ATPase activity of Rad54p is used to translocate the enzyme along the nucleosomal fiber, generating superhelical torsion, which leads to enhanced nucleosomal DNA accessibility. It seems likely that this chromatin remodeling activity of Rad54p might also facilitate additional steps after heteroduplex joint formation. Future studies are now poised to reconstitute the complete homologous recombinational repair reaction that fully mimics each step in the repair of chromosomal DNA double strand breaks in vivo (Jaskelioff, 2003).
In vivo and in vitro studies have suggested the following sequence of molecular events that lead to the recombinational repair of a DSB. First, the 5' ends of DNA that flank the break are resected by an exonuclease to create ssDNA tails. Next, Rad51p polymerizes onto these DNA tails to form a nucleoprotein filament that has the capability to search for a homologous duplex DNA molecule. After DNA homology has been located, the Rad51-ssDNA nucleoprotein filament catalyzes the formation of a heteroduplex DNA joint with the homolog. The process of DNA homology search and DNA joint molecule formation is called 'homologous DNA pairing and strand exchange'. Subsequent steps entail DNA synthesis to replace the missing information followed by resolution of DNA intermediates to yield two intact duplex DNA molecules (Jaskelioff, 2003).
The homologous DNA pairing activity of Rad51p is enhanced by Rad54p. Rad54p is a member of the Swi2p/Snf2p protein family that has DNA-stimulated ATPase activity and physically interacts with Rad51p. Because of its relatedness to the Swi2p/Snf2p family of ATPases, Rad54p may have chromatin remodeling activities in addition to its established role in facilitating Rad51p-mediated homologous pairing reactions. In this study it has been shown that Rad51p and Rad54p mediate robust D-loop formation with a chromatin donor, whereas the bacterial recombinase, RecA, can only function with naked DNA. Furthermore, the ATPase activity of Rad54p is essential for D-loop formation on chromatin and Rad54p can use the free energy from ATP hydrolysis to enhance the accessibility of nucleosomal DNA. Experiments are also presented to suggest that chromatin remodeling by Rad54p and yeast SWI/SNF involves DNA translocation (Jaskelioff, 2003).
Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, three distinct foci are observed that colocalize with adjacent GFP chromosome marks. The kinetics of focus formation with recombination intermediates and products were compared when HO-cleaved MATalpha recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. Evidence is presented for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52 (Miyazaki, 2004; full text of article).
The efficient and accurate repair of DNA double strand breaks (DSBs) is critical to cell survival, and defects in this process can lead to genome instability and cancers. In eukaryotes, the Rad52 group of proteins dictates the repair of DSBs by the error-free process of homologous recombination (HR). A critical step in eukaryotic HR is the formation of the initial Rad51-single-stranded DNA presynaptic nucleoprotein filament. This presynaptic filament participates in a homology search process that leads to the formation of a DNA joint molecule and recombinational repair of the DSB. The Rad54 protein functions as a mediator of Rad51 binding to single-stranded DNA; this activity does not require ATP hydrolysis. A novel Rad54-dependent chromatin remodeling event has been discovered that occurs in vivo during the DNA strand invasion step of HR. This ATP-dependent remodeling activity of Rad54 appears to control subsequent steps in the HR process (Wolner, 2005; full text of article).
Rad51, Rad52, and RPA play central roles in homologous DNA recombination. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 has two distinct activities: to recruit Rad51 onto single-strand (ss)DNA that is complexed with the ssDNA-binding protein, RPA, and to anneal complementary ssDNA complexed with RPA. This study reports that Rad52 promotes annealing of the ssDNA strand that is displaced by DNA strand exchange by Rad51 and RPA, to a second ssDNA strand. An RPA that is recombination-deficient (RPArfa1-t11) failed to support annealing, explaining its in vivo phenotype. Escherichia coli RecO and SSB proteins, which are functional homologues of Rad52 and RPA, also facilitate the same reaction, demonstrating its conserved nature. The two activities of Rad52, recruiting Rad51 and annealing DNA, are coordinated in DNA strand exchange and second ssDNA capture (Sugiyama, 2006).
Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, fluorescence resonance energy transfer (FRET) and immune-precipitation experiments were used to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. The WRN-RAD51 and WRN-RAD54B direct interaction was verified in vitro. These data support a role for WRN also in the recombination step of ICL repair (Otterlei, 2006).
Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1- and BRCA2-mediated tumor suppression as well as cellular viability, a functional analysis of a defined RAD51 mutation was performed in mammalian cells. By using a dominant negative approach, a mouse embryonic stem cell line was generated that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. It was found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, it was also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells (Stark, 2002; full text of article).
Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. The current results show that Ca(2+) greatly stimulates DNA strand exchange activity of hRad51 protein. Ca(2+) exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. The data demonstrate that, in the presence of Mg(2+), the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca(2+) maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; no such mechanism was found for yeast Rad51 (yRad51) protein (Bugreev, 2004).
RecA in Escherichia coli and its homolog, ScRad51 in Saccharomyces cerevisiae, are known to be essential for recombinational repair. The homolog of RecA and ScRad51 in mice, MmRad51, was mutated to determine its function. Mutant embryos arrest early during development. A decrease in cell proliferation, followed by programmed cell death and chromosome loss, was observed. Radiation sensitivity was demonstrated in trophectoderm-derived cells. Interestingly, embryonic development progressed further in a p53 null background; however, fibroblasts derived from double-mutant embryos failed to proliferate in tissue culture (Lim, 1996).
The mouse Rad51 gene is a mammalian homolog of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cells. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability (Tsuzuki, 1996).
The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. It is suggested that the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding (Fung, 2006).
Genomic instability is characteristic of tumor cells, and a strong correlation exists between abnormal karyotype and tumorigenicity. Increased expression of the homologous recombination and DNA repair protein Rad51 has been reported in immortalized and tumor cells, which could alter recombination pathways to contribute to the chromosomal rearrangements found in these cells. A genetic system was used to examine the potential for multiple double-strand breaks to lead to genome rearrangements in the presence of increased Rad51 expression. Analysis of repair revealed a novel class of products consistent with crossing over, involving gene conversion associated with an exchange of flanking markers leading to chromosomal translocations. Increased Rad51 also promoted aneuploidy and multiple chromosomal rearrangements. These data provide a link between elevated Rad51 protein levels, genome instability, and tumor progression (Richardson, 2004).
The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells (Liu, 1998).
DMC1 is a meiosis-specific gene first discovered in yeast that encodes a protein with homology to RecA and may be a component of recombination nodules. Yeast dmc1 mutants are defective in crossing over and synaptonemal complex (SC) formation, and arrest in late prophase of meiosis I. A null mutation was generated in the Dmc1 gene in mice; homozygous mutant males and females are sterile with arrest of gametogenesis in the first meiotic prophase. Chromosomes in mutant spermatocytes fail to synapse, despite the formation of axial elements that are the precursor to the SC. The strong similarity of phenotypes in Dmc1-deficient mice and yeast suggests that meiotic mechanisms have been highly conserved through evolution (Pittman, 1998).
The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and Rad51C-Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, homology modeling was used to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, it has been demonstrated that a fragment of Rad51B containing amino acid residues 1-75 interacts with the C-terminus and linker of Rad51C, residues 79-376, and this region of Rad51C also interacts with mRad51D and Xrcc3. It has also been determined that the N-terminal domain of mRad51D, residues 4-77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77-328, binds Rad51C. By this, the binding domains of the BCDX2 and CX3 complexes have been demonstrated, the interaction of these proteins has been further demonstrated, and a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes is proposed (Miller, 2004).
During genetic recombination and the recombinational repair of chromosome breaks, DNA molecules become linked at points of strand exchange. Branch migration and resolution of these crossovers, or Holliday junctions (HJs), complete the recombination process. This study shows that extracts from cells carrying mutations in the recombination/repair genes RAD51C or XRCC3 have reduced levels of HJ resolvase activity. Moreover, depletion of RAD51C from fractionated human extracts caused a loss of branch migration and resolution activity, but these functions were restored by complementation with a variety of RAD51 paralog complexes containing RAD51C. It is concluded that the RAD51 paralogs are involved in HJ processing in human cells (Liu, 2004a).
Programmed double-strand breaks at prophase of meiosis acquire immunologically detectable RAD51-DMC1 foci or early nodules (ENs) that are associated with developing chromosome core segments; each focus is surrounded by a gammaH2AX-modified chromosome domain. The 250-300 ENs per nucleus decline in numbers during the development of full-length cores and the remaining foci are relatively evenly distributed along the mature cores. The ENs become transformed nodules (TNs) by the acquisition of RPA, BLM, MSH4 and topoisomerases that function in repair and Holliday junction resolution. At the leptotene-zygotene transition, TNs orient to positions between the aligned cores where they initiate structural interhomolog contacts prior to synaptonemal complex (SC) formation, possibly future crossover sites. Subsequently, TNs are associated with SC extension at the synaptic forks. Dephosphorylation of TN-associated histone gammaH2AX chromatin suggests annealing of single strands or repair of double-strand breaks DSBs at this time. Some 200 TNs per pachytene nucleus are distributed proportional to SC length and are evenly distributed along the SCs. At this stage, gammaH2AX-modified chromatin domains are associated with transcriptionally silenced sex chromosomes and autosomal sites. Immunogold electron microscope evidence shows that one or two TNs of the 10-15 TNs per SC acquire MLH1 protein, the hallmark of reciprocal recombination, whereas the TNs that do not acquire MLH1 protein relocate from their positions along the midline of the SCs to the periphery of the SCs. Relocation of TNs may be associated with the conversion of potential crossovers into non-crossovers (Moens, 2007).
RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, a hypomorphic allele of Rad51c was generated in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, it is proposed that this late function may be associated with HJ resolution (Kuznetsov, 2007).
In germ line cells, recombination is required for gene reassortment and proper chromosome segregation at meiosis, whereas in somatic cells it provides an important mechanism for the repair of DNA double-strand breaks. Five proteins (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) that share homology with RAD51 recombinase and are known as the RAD51 paralogs are important for recombinational repair; paralog-defective cell lines exhibit spontaneous chromosomal aberrations, defective DNA repair, and reduced gene targeting. The paralogs form two distinct protein complexes, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3, but their precise cellular roles remain unknown. This study shows that, like MLH1, RAD51C localizes to mouse meiotic chromosomes at pachytene/diplotene. Using immunoprecipitation and gel filtration analyses, it was found that Holliday junction resolvase activity associates tightly and co-eluted with the 80-kDa RAD51C-XRCC3 complex. Taken together, these data indicate that the RAD51C-XRCC3-associated Holliday junction resolvase complex associates with crossovers and may play an essential role in the resolution of recombination intermediates prior to chromosome segregation (Liu, 2007).
Members of the RecQ helicase family play critical roles in genome maintenance. There are five RecQ homologs in mammals, and defects in three of these (BLM, WRN, and RECQL4) give rise to cancer predisposition syndromes in humans. RECQL and RECQL5 have not been associated with a human disease. This study shows that deletion of Recql5 in mice results in cancer susceptibility. Recql5-deficient cells exhibit elevated frequencies of spontaneous DNA double-strand breaks and homologous recombination (HR) as scored using a reporter that harbors a direct repeat, and are prone to gross chromosomal rearrangements in response to replication stress. To understand how RECQL5 regulates HR, purified proteins were used to demonstrate that human RECQL5 binds the Rad51 recombinase and inhibits Rad51-mediated D-loop formation. By biochemical means and electron microscopy, it was shown that RECQL5 displaces Rad51 from single-stranded DNA (ssDNA) in a reaction that requires ATP hydrolysis and RPA. Together, these results identify RECQL5 as an important tumor suppressor that may act by preventing inappropriate HR events via Rad51 presynaptic filament disruption (Hu, 2007).
Homologous recombination (HR) is a fundamental molecular process in all organisms. In meiosis, HR is necessary for the proper segregation of homologous chromosomes and generates genetic diversity through the shuffling of parental alleles. In mitotic cells, HR is an important pathway for repairing chromosomal breaks and gaps, and for restarting damaged or stalled DNA replication forks. However, inappropriate or untimely HR events can have mutagenic and oncogenic consequences. For example, reciprocal exchanges (crossovers) between homologous chromosomes can lead to somatic loss of heterozygosity (LOH), while crossovers between nonhomologous chromosomes can result in translocation. Also, crossovers between repeated sequences on the same chromosome can result in deletions or inversions. For these reasons, specific mechanisms have evolved for regulating HR to minimize these potentially deleterious rearrangements (Hu, 2007).
Genetic analyses in Saccharomyces cerevisiae indicate that two DNA helicases, Srs2 and Sgs1, function in different pathways to suppress crossover events in mitotic cells. Mutations in either Srs2 or Sgs1 result in a hyperrecombination phenotype. In humans, mutations in BLM, which encodes the human ortholog of Sgs1, give rise to the rare hereditary disorder Bloom's syndrome. This disorder is marked by an elevated rate of sister chromatid exchange (SCE), increased chromosomal instability, and a high incidence of cancer. BLM suppresses SCEs by acting in conjunction with the Type 1A topoisomerase, Topo IIIα, and a recently identified protein, BLAP75, to mediate the dissolution of double Holliday junctions (DHJ; a late HR intermediate), a process that yields solely noncrossover recombinants. Like its human counterpart, Sgs1 forms a complex with the Top3 topoisomerase and Rmi1 (the BLAP75 ortholog), suggesting that it might function to suppress SCEs by a similar mechanism (Hu, 2007).
Srs2 is a superfamily 1 helicase with similarities to the bacterial UvrD/Rep helicases. The mechanistic basis of the Srs2 function was elucidated by biochemical studies that revealed its ability to bind Rad51 and to dismantle the Rad51-ssDNA (single-stranded DNA) nucleoprotein filament, the key catalytic intermediate in recombination reactions. To date, no apparent Srs2 ortholog has been identified in other eukaryotes, although the recently identified Fbh1 helicase shows some structural similarity to the Srs2 helicase family and studies in Schizosaccharomyces pombe suggest that Fbh1 plays a role in processing HR intermediates. However, Fbh1-deficient DT40 cells show no prominent sensitivity to DNA damaging agents, and exhibit only a mild SCE phenotype. This raises the question as to whether attenuation of HR by disruption of the Rad51 presynaptic filament represents a significant mechanism for HR regulation in higher eukaryotes (Hu, 2007).
Sgs1 and BLM are members of the RecQ family of DNA helicases. Sgs1 is the sole RecQ helicase in budding yeast. Interestingly, humans have a total of five RecQ helicase encoding genes (RECQL, BLM, WRN, RECQL4, and RECQL5). They share a conserved seven-motif helicase domain but are otherwise distinct from one another by their unique amino acid composition outside the helicase domain, suggesting that they have related but different roles. In addition to BLM, defects in both WRN and RECQL4 are also associated with heritable genome instability and cancer disorders. Therefore, while BLM likely represents the Sgs1 ortholog, the other RECQ-like helicases represent potential candidates as the functional equivalent of Srs2 in humans. Mouse cells deficient in the RECQL5 homolog Recql5 exhibit an elevated level of SCEs, thus implicating this helicase in the regulation of HR. Importantly, deletion of both Recql5 and Blm further increases the SCE frequency, consistent with Recql5 acting to regulate SCEs in mitotic cells via a mechanism that is distinct from Blm, perhaps by functioning similarly to Srs2 to suppress the channeling of DNA lesions into HR (Hu, 2007).
This paper shows that deletion of Recql5 in mice results in increased susceptibility to cancer. Recql5-deficient cells exhibit elevated frequencies of spontaneous double-strand breaks (DSBs) and HR between direct repeats, and are prone to the accumulation of gross chromosomal rearrangements (GCRs) in response to replication stress. Moreover, by biochemical means, a mechanistic basis by which RECQL5 functions in suppressing GCRs and tumorigenesis is provided. Specifically, human RECQL5 binds the Rad51 recombinase, and a catalytic quantity of this helicase inhibits Rad51-mediated D-loop formation markedly. Furthermore, RECQL5 displaces Rad51 from ssDNA in a reaction that requires ATP hydrolysis by RECQL5 and is stimulated by the ssDNA-binding protein RPA. Taken together, these data provide compelling evidence that this unique member of the RecQ helicase family functions to minimize the propensity of oncogenic rearrangements by suppressing the accumulation of DSBs and attenuating HR by disrupting the Rad51 presynaptic filament (Hu, 2007).
The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. This study identifies a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, it was found that a specific mcm (see Drosophila Mcm2) allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX (see Drosophila His2Av) and Rad52 (see Drosophila spn-A). Further, the triggering events for this genome instability were found to be elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1 (see Drosophila mei-41), suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, the study suggests that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed (Vijayraghavan, 2016).
Search PubMed for articles about Drosophila Rad51-like
Abdu, U., Brodsky, M. and Schüpbach, T. (2002). Activation of a meiotic checkpoint during Drosophila oogenesis regulates the translation of Gurken through Chk2/Mnk. Curr. Biol. 12: 1645-1651. 12361566
Abdu, U., Gonzalez-Reyes, A., Ghabrial, A. and Schüpbach, T. (2003). The Drosophila spn-D gene encodes a RAD51C-like protein that is required exclusively during meiosis. Genetics, 165: 197-204. 14504227
Abdu, U., Klovstad, M., Butin-Israeli, V., Bakhrat, A. and Schupbach, T. (2007). An essential role for Drosophila hus1 in somatic and meiotic DNA damage responses. J. Cell Sci. 120: 10421049. PubMed Citation: 17327271
Adams, M. D., McVey, M. and Sekelsky, J. J. (2003). Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing. Science 299: 265-267. 12522255
Akamatsu, Y., et al. (2007). Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51-dependent recombination outcomes. EMBO J. 26(5): 1352-62. Medline abstract: 17304215
Alexeev, A., Mazin, A. and Kowalczykowski, S. C. (2003). Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament. Nat. Struct. Biol. 10(3): 182-6. 12577053
Alexiadis, V. and Kadonaga, J. T. (2002). Strand pairing by Rad54 and Rad51 is enhanced by chromatin. Genes Dev. 16: 2767-2771. 12414729
Baumann, P., Benson, F. E., and West, S. C. (1996). Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87: 757-766. 8929543
Beaucher, M., Zheng, X. F., Amariei, F. and Rong, Y. S. (2012). Multiple pathways suppress telomere addition to DNA breaks in the Drosophila germline. Genetics 191: 407-417. PubMed ID: 22446318
Benson, F. E., Baumann, P. and West, S. C. (1998). Synergistic actions of Rad51 and Rad52 in recombination and DNA repair. Nature 391(6665): 401-404.
Bishop, D. K., Park, D., Xu, L. and Kleckner, N. (1992). DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation and cell cycle progression. Cell 69: 439-456. 1581960
Bishop, D. K., Nikolski, Y., Oshiro, J., Chon, J., Shinohara, M. and Chen, X. (1999). High copy number suppression of the meiotic arrest caused by a dmc1 mutation: REC114 imposes an early recombination block and RAD54 promotes a DMC1-independent DSB repair pathway. Genes Cells 4: 425-444. 10526232
Brenneman, M. A., Wagener, B. M., Miller, C. A., Allen, C. and Nickoloff, J. A. (2002). XRCC3 controls the fidelity of homologous recombination: roles for XRCC3 in late stages of recombination. Mol. Cell 10: 387-395. 12191483
Brough, R., Wei, D., Leulier, S., Lord, C. J., Rong, Y. S. and Ashworth, A. (2008). Functional analysis of Drosophila melanogaster BRCA2 in DNA repair. DNA Repair (Amst) 7: 1019. PubMed Citation: 17822964
Bugreev, D. V. and Mazin, A. V. (2004). Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc. Natl. Acad. Sci. 101: 9988-9993. Medline abstract: 15226506
Chen, F., et al. (1997). Cell cycle-dependent protein expression of mammalian homologs of yeast DNA double-strand break repair genes Rad51 and Rad52. Mutat. Res. 384(3): 205-211.
Chen Y, Pane A, Schupbach T (2007) Cutoff and aubergine mutations result in retrotransposon upregulation and checkpoint activation in Drosophila. Curr. Biol. 17: 637642. PubMed Citation: 17363252
Chi, P., et al. (2006). Yeast recombination factor Rdh54 functionally interacts with the Rad51 recombinase and catalyzes Rad51 removal from DNA. J. Biol. Chem. 281: 26268-26279. Medline abstract: 16831867
Clark, A. J. (1996). recA mutants of E. coli K12: a personal turning point. Bioessays 18(9): 767-72. 8831293
Clever B., et al. (1997). Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins. EMBO J. 16(9): 2535-2544.
Davies, O. R. and Pellegrini, L. (2007). Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats. Nat. Struct. Mol. Biol. 14: 475483. PubMed Citation: 17515903
Davis, A. P. and Symington, L. S. (2004). RAD51-dependent break-induced replication in yeast. Mol. Cell Biol. 24(6): 2344-51. 14993274
Dekanty, A., Barrio, L. and Milan, M. (2014). Contributions of DNA repair, cell cycle checkpoints and cell death to suppressing the DNA damage-induced tumorigenic behavior of Drosophila epithelial cells. Oncogene [Epub ahead of print]. PubMed ID: 24632609
Delacroix, S., et al. (2007). The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev. 21: 14721477. PubMed Citation: 17575048
Esashi, F., Galkin, V. E., Yu, X., Egelman, E. H. and West, S. C. (2007). Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2. Nat. Struct. Mol. Biol. 14:468474. PubMed Citation: 17515904
Fortin, G. S., Symington, L. S. (2002). Mutations in yeast Rad51 that partially bypass the requirement for Rad55 and Rad57 in DNA repair by increasing the stability of Rad51-DNA complexes. EMBO J. 21(12): 3160-70. 12065428
Fung, C. W., et al. (2006). The rad51-K191R ATPase-defective mutant is impaired for presynaptic filament formation. Mol. Cell. Biol. 26(24): 9544-54. Medline abstract: 17030607
Gao, G., Bi, X., Chen, J., Srikanta, D. and Rong, Y. S. (2009). Mre11-Rad50-Nbs complex is required to cap telomeres during Drosophila embryogenesis. Proc Natl Acad Sci U S A 106: 10728-10733. PubMed ID: 19520832
Ghabrial, A., Ray, R. P. and Schupbach, T. (1998). okra and spindle-B encode components of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila oogenesis. Genes Dev. 12: 2711-2723. 9732269
Ghabrial, A. and Schüpbach, T. (1999). Activation of a meiotic checkpoint regulates translation of Gurken during Drosophila oogenesis. Nat. Cell Biol. 1, 354-357. 10559962
Gonzalez-Reyes, A., Elliott, H. and St Johnston, D. (1997). Oocyte determination and the origin of polarity in Drosophila: the role of the spindle genes. Development 124: 4927-4937. 9362456
Holzen, T. M., et al. (2006). Tid1/Rdh54 promotes dissociation of Dmc1 from nonrecombinogenic sites on meiotic chromatin. Genes Dev. 20: 2593-2604. Medline abstract: 16980587
Hong, E. L., Shinohara, A. and Bishop, D. K. (2001). Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA. J. Biol. Chem. 276(45): 41906-12. 11551925
Hu, Y., et al. (2007). RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments. Genes Dev. 21: 3073-3084. PubMed Citation: 18003859
Huynh, J. R. and St Johnston, D. (2000). The role of BicD, Egl, Orb and the microtubules in the restriction of meiosis to the Drosophila oocyte. Development, 127: 2785-2794. 10851125
Jang, J. K., Sherizen, D. E., Bhagat, R., Manheim, E. A. and McKim, K. S. (2003). Relationship of DNA double-strand breaks to synapsis in Drosophila. J. Cell Sci. 116: 3069-3077. 12799415
Jaskelioff, M., et al. (2003). Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin. J. Biol. Chem. 278: 9212-9218. 12514177
Jiang, H., et al. (1996). Direct association between the yeast Rad51 and Rad54 recombination proteins. J. Biol. Chem. 271(52): 33181-33186.
Johnson-Schlitz, D. and Engels, W. R. (2006). Template disruptions and failure of double Holliday junction dissolution during double-strand break repair in Drosophila BLM mutants. Proc. Natl. Acad. Sci. 103(45): 16840-5. Medline abstract: 17075047
Johnson-Schlitz, D. M., Flores, C. and Engels, W. R. (2007). Multiple-pathway analysis of double-strand break repair mutations in Drosophila. PLoS Genet. 3(4): e50. PubMed Citation: 17432935
Kiianitsa, K., Solinger, J. A. and Heyer, W.-D. (2002). Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein. J. Biol. Chem. 277: 46205-46215. 12359723
Klovstad, M., Abdu, U. and Schüpbach, T. (2008). Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint. PLoS Genet. 4(2): e31. PubMed Citation: 18266476
Kraakman-van der Zwet, M., et al. (2002). Brca2 (XRCC11) deficiency results in radioresistant DNA synthesis and a higher frequency of spontaneous deletions. Mol. Cell. Biol. 22: 669679. PubMed Citation: 11756561
Krejci, L., et al. (2003). DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423: 305-309. Medline abstract: 12748644
Kuznetsov, S., et al. (2007). RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females. J. Cell Biol. 176(5): 581-92. Medline abstract: 17312021
Larminat, F., Germanier, M., Papouli, E. and Defais, M. (2002). Deficiency in BRCA2 leads to increase in non-conservative homologous recombination. Oncogene 21: 51885192. PubMed Citation: 12140769
Lee, S. E., et al. (2003). Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break. Mol. Cell Biol. 23(23): 8913-23. 14612428
Lettier, G., et al. (2006). The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae. PLoS Genet. 2(11): e194. Medline abstract: 17096599
Liberi, G., et al. (2005). Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev. 19(3): 339-50. 15687257
Lim, D. S. and Hasty, P. (1996). A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol. Cell. Biol. 16: 7133-7143. 8943369
Lin, H. R., Ting, N. S., Qin, J. and Lee, W. H. (2003). M phase-specific phosphorylation of BRCA2 by Polo-like kinase 1 correlates with the dissociation of the BRCA2-P/CAF complex. J. Biol. Chem. 278(38): 35979-87. 12815053
Liu, N. et al. (1998). XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. Mol. Cell 1: 783-793. 9660962
Liu, Y., et al. (2004a). RAD51C is required for Holliday junction processing in mammalian cells. Science 303: 243-246. Medline abstract: 14716019
Liu, Y., et al. (200b4). Conformational changes modulate the activity of human RAD51 protein. J. Mol. Biol. 337(4): 817-27. 15033353
Liu, Y., Tarsounas, M., O'regan, P. and West, S. C. (2007). Role of RAD51C and XRCC3 in genetic recombination and DNA repair. J. Biol. Chem. 282(3): 1973-9. Medline abstract: 17114795
Lo, T., Pellegrini, L., Venkitaraman, A. R. and Blundell, T. L. (2003). Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer. DNA Repair 2: 10151028. PubMed Citation: 12967658
Lomonosov, M., Anand, S., Sangrithi, M., Davies, R. and Venkitaraman, A. R. (2003). Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein. Genes Dev. 17: 30173022. PubMed Citation: 14681210
Ludwig, T., Chapman, D. L., Papaioannou, V. E. and Efstratiadis, A. (1997). Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos. Genes Dev 11: 12261241. PubMed Citation: 9171368
Madigan, J. P., Chotkowski, H. L. and Glaser, R. L. (2002). DNA double-strand break-induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis. Nucleic Acids Res. 30: 3698-3705. 12202754
Martin, J. S., Winkelmann, N., Petalcorin, M. I., McIlwraith, M. J. and Boulton, S. J. (2005). RAD-51-dependent and -independent roles of a Caenorhabditis elegans BRCA2-related protein during DNA double-strand break repair. Mol Cell Biol 25: 31273139. PubMed Citation: 15798199
Mazin, A. V., Zaitseva, E., Sung, P. and Kowalczykowski, S. C. (2000a). Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing. EMBO J. 19: 1148-1156. 10698955
Mazin, A. V., Bornarth, C. J., Solinger, J. A., Heyer, W.-D. and Kowalczykowski, S.C. (2000b). Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament. Mol. Cell 6: 583-592. 11030338
Mazin, A. V., Alexeev, A. A. and Kowalczykowski, S. C. (2003). A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J. Biol. Chem. 278(16): 14029-36. 12566442
Mazina, O. M., et al. (2004). Saccharomyces cerevisiae Mer3 helicase stimulates 3'-5' heteroduplex extension by Rad51: implications for crossover control in meiotic recombination. Cell 117: 47-56. 15066281
McKee, B. D., Ren, X. and Hong, C. (1996) A recA-like gene in Drosophila melanogaster that is expressed at high levels in female but not male meiotic tissues. Chromosoma 104: 479-488. 8625736
Miller, K. A., Sawicka, D., Barsky, D. and Albala, J. S. (2004). Domain mapping of the Rad51 paralog protein complexes. Nucleic Acids Res. 32(1): 169-78. 14704354
Miyazaki, T., Bressan, D. A., Shinohara, M., Haber, J. E. and Shinohara, A. (2004). In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J. 23(4): 939-49. 14765116
Moens, P. B., et al. (2007). Initiation and resolution of interhomolog connections: crossover and non-crossover sites along mouse synaptonemal complexes. J. Cell Sci. 120(Pt 6): 1017-27. Medline abstract: 17344431
Morris, J. and Lehmann, R. (1999). Drosophila oogenesis: versatile spn doctors. Curr. Biol. 9: R55-R58. 10021357
Muris, D. F. R., et al. (1997). Homologous recombination in the fission yeast Schizosaccharomyces pombe: different requirements for the rhp51+, rhp54+ and rad22+ genes. Curr. Genet. 31(3): 248-254.
New, J. H., et al. (1998). Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 391(6665): 407-410.
Otterlei M., et al. (2006). Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest. J. Cell Sci. 119(Pt 24): 5137-46. Medline abstract: 17118963
Paques, F. and Haber, J. E. (1999). Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63: 349404. PubMed Citation: 10357855
Patel, K. J., et al. (1998). Involvement of Brca2 in DNA repair. Mol. Cell 1: 347357. PubMed Citation: 9660919
Petukhova, G., Stratton, S. and Sung, P. (1998). Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393(6680): 91-94.
Petukhova, G., Van Komen, S., Vergano, S., Klein, H., and Sung, P. (1999). Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation. J. Biol. Chem. 274: 29453-29462. 10506208
Pittman, D. L., Cobb, J., Schimenti, K. J., Wilson, L. A., Cooper, D. M., Brignull, E., Handel, M. A. and Schimenti, J. C. (1998). Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog. Mol. Cell 1: 697-705. 9660953
Prakash, R., et al. (2009). Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Genes Dev. 23(1): 67-79. PubMed Citation: 19136626
Preston, C. R., Flores, C. C. and Engels, W. R. (2006). Differential usage of alternative pathways of double-strand break repair in Drosophila. Genetics 172: 10551068. PubMed Citation: 16299390
Rice, K. P., Eggler, A. L., Sung, P. and Cox, M. M. (2001). DNA pairing and strand exchange by the Escherichia coli RecA and yeast Rad51 proteins without ATP hydrolysis: on the importance of not getting stuck. J. Biol. Chem. 276(42): 38570-81. 11504729
Richardson, C., Stark, J. M., Ommundsen, M. and Jasin, M. (2004). Rad51 overexpression promotes alternative double-strand break repair pathways and genome instability. Oncogene 23(2): 546-53. 14724582
Ristic, D., et al. (2005). Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res. 33: 3292-3302. Medline abstract: 15944450
Roeder, G. S. (1997). Meiotic chromosomes: it takes two to tango. Genes Dev. 11: 2600-2621. 9334324
Roeder, G. S. and Bailis, J. M. (2000). The pachytene checkpoint. Trends Genet. 16: 395-403. 10973068
Sekelsky, J. J., Brodsky, M. H., Burtis, K. C. (2000). DNA repair in Drosophila: insights from the Drosophila genome sequence. J. Cell Biol. 150(2): F31-F36. 10908583
Shen, W. H., et al. (2007). Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell 128(1): 157-70. Medline abstract: 17218262
Shin, D. S., et al. (2003). Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. EMBO J. 22(17): 4566-76. 12941707
Shinohara, A., Ogawa, H. and Ogawa, T. (1992). Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69: 457-470. 1581961
Shinohara, A. and Ogawa, T. (1998). Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391(6665): 404-407.
Sigurdsson, S., Trujillo, K., Song, B., Stratton, S. and Sung, P. (2001). Basis for avid homologous DNA strand exchange by human Rad51 and RPA J. Biol. Chem. 276: 8798-8806. 11124265
Sigurdsson, S., Van Komen, S., Petukhova, G. and Sung, P. (2002). Homologous DNA pairing by human recombination factors Rad51 and Rad54. J. Biol. Chem. 277(45): 42790-4. 12205100
Sinha, M., et al. (2009). Recombinational repair within heterochromatin requires ATP-dependent chromatin remodeling. Cell 138(6): 1109-21. PubMed Citation: 19766565
Solinger, J. A., Lutz, G., Sugiyama, T., Kowalczykowski, S. C. and Heyer, W.-D. (2001a). Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament. J. Mol. Biol. 307: 1207-1221. 11292336
Solinger, J. A. and Heyer, W.-D. (2001b). Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange. Proc. Natl. Acad. Sci. 98: 8447-8453. 11459988
Sonoda, E., Sasaki, M.S., Buerstedde, J. M., Bezzubova, O., Shinohara, A., Ogawa, H., Takata, M., Yamaguchi-Iwai, Y. and Takeda, S. (1998). Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17: 598-608. 9430650
Spell, R. M. and Jinks-Robertson, S. (2003). Role of mismatch repair in the fidelity of RAD51- and RAD59-dependent recombination in Saccharomyces cerevisiae. Genetics 165(4): 1733-44. 14704162
Staeva-Vieira, E., Yoo, S. and Lehmann, R. (2003). An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control. EMBO J. 22: 5863-5874. 14592983
Stark, J. M., et al. (2002). ATP hydrolysis by mammalian RAD51 has a key role during homology-directed DNA repair. J. Biol. Chem. 277: 20185-20194. Medline abstract: 11923292
Stark, J. M., Pierce, A. J., Oh, J., Pastink, A. and Jasin, M. (2004). Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol. Cell Biol. 24: 93059316. PubMed Citation: 15485900
Styhler, S., Nakamura, A., Swan, A., Suter, B. and Lasko, P. (1998). vasa is required for GURKEN accumulation in the oocyte and is involved in oocyte differentiation and germline cyst development. Development, 125: 1569-1578
Sugawara, N., Wang, X. and Haber, J. E. (2003). In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Molec. Cell 12: 209-219. 12887906
Sugiyama, T., et al. (2006). Rad52-mediated DNA annealing after Rad51-mediated DNA strand exchange promotes second ssDNA capture. EMBO J. 25(23): 5539-48. Medline abstract: 17093500
Sung, P., Krejci, L., Van Komen, S. and Sehorn, M. G. (2003). Rad51 recombinase and recombination mediators. J. Biol. Chem. 278(44): 42729-32. 12912992
Tarsounas, M., et al. (2004). Telomere maintenance requires the RAD51D recombination/repair protein. Cell 117: 337-347. 15109494
Tashiro, S., Walter, J., Shinohara, A., Kamada, N. and Cremer, T. (2000). Rad51 accumulation at sites of DNA damage and in postreplicative chromatin. J. Cell Biol. 150(2): 283-91. 10908572
Tearle, R. and Nusslein-Volhard, C. (1987). Tubingen mutants and stock list. Drosophila Inform. Serv. 66: 209-226.
Terasawa M., et al. (2007). Meiotic recombination-related DNA synthesis and its implications for cross-over and non-cross-over recombinant formation. Proc. Natl. Acad. Sci. 104(14): 5965-70. Medline abstract: 17384152
Tomancak, P., Guichet, A., Zavorszky, P. and Ephrussi, A. (1998). Oocyte polarity depends on regulation of gurken by Vasa. Development 125: 1723-1732. 9521910
Trojanek, J., et al. (2003). Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination. Mol. Cell. Biol. 23(21): 7510-24. 14559999
Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y. and Morita, T. (1996). Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl Acad. Sci. 93: 6236-6240. 8692798
Van Komen, S., Petukhova, G., Sigurdsson, S., Stratton, S., and Sung, P. (2000). Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol. Cell 6: 563-572. 11030336
Van Komen, S., Petukhova, G., Sigurdsson, S. and Sung, P. (2002). Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation. J. Biol. Chem. 277(46): 43578-87. 1222608
Veaute, X., et al. (2003). The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423: 309-312. Medline abstract: 12748645
Venkitaraman, A. R. (2002). Cancer Susceptibility and the Functions of BRCA1 and BRCA2. Cell 108: 171-182. PubMed Citation: 11832208
Vijayraghavan, S., Tsai, F.L. and Schwacha, A. (2016). A checkpoint-related function of the MCM replicative helicase is required to avert accumulation of RNA:DNA hybrids during S-phase and ensuing DSBs during G2/M. PLoS Genet 12: e1006277. PubMed ID: 27556397
Wolner, B., and Peterson, C. L. (2005). ATP-dependent and ATP-independent roles for the Rad54 chromatin remodeling enzyme during recombinational repair of a DNA double strand break. J. Biol. Chem. 280: 10855-10860. Medline abstract: 15653683
Yamada, N. A., Hinz, J. M., Kopf, V. L., Segalle, K. D. and Thompson, L. H. (2004). XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination. J. Biol. Chem. 279(22): 23250-4. 15037616
Yang, H., Li, Q., Fan, J., Holloman, W. K. and Pavletich, N. P. (2005). The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction. Nature 433: 653657. PubMed Citation: 15703751
Yang, Y. G., et al. (2006). Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks. EMBO J. 25(23): 5527-38. Medline abstract: 17082765
Yoo, S. (2006). Characterization of Drosophila Rad51/SpnA protein in DNA binding and embryonic development. Biochem. Biophys. Res. Commun. 348(4): 1310-8. Medline abstract: 16919604
Yu, D. S., et al. (2003). Dynamic control of Rad51 recombinase by self-association and interaction with BRCA2. Mol. Cell 12(4): 1029-41. 14580352
Yu, V. P., et al. (2000). Gross chromosomal rearrangements and genetic exchange between nonhomologous chromosomes following BRCA2 inactivation. Genes Dev. 14: 14001406. PubMed Citation: 10837032
Zaitseva, E. M., Zaitsev, E. N., and Kowalczykowski, S. C. (1999). The DNA binding properties of Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 274: 2907-2915. 9915828
Zhao, G. Y., et al. (2007). A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol. Cell 25(5): 663-75. Medline abstract: 17349954
date revised: 20 October 2016
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