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).
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