Breast cancer 2, early onset homolog: Biological Overview | References
Gene name - Breast cancer 2, early onset homolog
Cytological map position - 60D4-60D4
Function - signaling
Keywords - DNA damage response, homologous recombination, transduction of meiotic recombination checkpoint signal
Symbol - Brca2
FlyBase ID: FBgn0050169
Genetic map position - 2R: 20,413,723..20,417,104 [+]
Classification - BRC repeats protein
Cellular location - nuclear
|Recent literature||Kocak, E., Dykstra, S., Nemeth, A., Coughlin, C. G., Rodgers, K. and McVey, M. (2019). The Drosophila melanogaster PIF1 helicase promotes survival during replication stress and processive DNA synthesis during double-strand gap repair. Genetics. PubMed ID: 31537623
PIF1 is a 5' to 3' DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, pif1 null mutants were created in Drosophila. pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, were found to experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.
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 to 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).
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).
The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Drosophila hus1 has been shown to be essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. This study further analyzed the function of hus1 during meiosis. The synaptonemal complex (SC) was found to disassemble abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, in hus1, DSBs that form during meiosis are not processed efficiently, and this defect is not suppressed by a mutation in DmChk2. A genetic interaction was found between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, these results imply that hus1 is required for repair of DSBs during meiotic recombination (Peretz, 2009)
When the integrity of the genetic material is compromised, the cell activates checkpoints that inhibit cell cycle progression, allowing for repair of the damaged DNA or, if unsuccessful, lead to cell death. The DNA damage checkpoint response involves a signal transduction pathway consisting of sensors, transducers and effectors. Hus1, Rad1 and Rad9 and the associated protein, Rad17 are thought to act as a sensor complex. The signal is transduced by ATM and ATM-Rad3-related (ATR) proteins along with Chk1 and Chk2 kinases. A wide range of effector proteins influence cellular fate following the DNA damage, among these are cell cycle arrest, apoptosis or activation of the DNA repair machinery. Various checkpoints exist, with each addressing a different type of DNA damage through the use of a specific set of signal transduction proteins (Peretz, 2009)
A meiotic recombination checkpoint, also known as the 'pachytene checkpoint' has been characterized in yeast. Meiotic recombination initiates with the generation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease. These breaks are repaired via homologous strand exchange with sequences on a non-sister chromatid. A set of proteins monitors recombination and activates a checkpoint during late prophase I (pachytene) if the recombination repair process has not been completed. This checkpoint prevents segregation of homologous chromosomes until recombination is complete and ensures proper distribution of the genetic material to the gametes (Peretz, 2009)
A meiotic checkpoint similar to that described in yeast also exists in Drosophila. Several of the spindle class genes were previously found to encode proteins with homology to known DNA repair enzymes. Specifically, spindle-A (spn-A) encodes a Rad51-like protein, spindle-B (spn-B) encodes a XRCC3-like protein, spindle-C (spn-C) encodes a HEL308-like protein, spindle-D (spn-D) encodes a Rad51C-like protein and okra encodes a Rad54-like protein. These genes were shown to be required for the repair of recombination-induced DSBs during Drosophila oogenesis. Moreover, mutations in these genes lead to activation of a meiotic checkpoint, leading to the appearance of several defects during oogenesis. The most obvious phenotypes manifested are the dorsal-ventral (D-V) patterning defects of the egg, arising due to improper localization and translation of gurken mRNA. In addition, the hollow sphere of highly packed chromatin (also called the karyosome) that is characteristic of the wild-type oocyte nucleus is often fragmented or thread-like in appearance in the DNA repair enzyme mutants. These defects can be suppressed by blocking the formation of DSBs during meiosis through mutations in the spo11 homologue, mei-W68, or by eliminating the checkpoint through a mutations in mei-41 or DmChk2, the Drosophila homologues for ATR and Chk2, respectively (Peretz, 2009)
hus1 mutant flies have been shown to be viable although the females are sterile. hus1 mutant flies are sensitive to hydroxyurea (HU) and to methyl methanesulfonate (MMS) but not to X-rays, suggesting that hus1 is required for the activation of an S phase checkpoint. Furthermore, hus1 is not required for the G2/M checkpoint or for post-irradiation induction of apoptosis. hus1 is able to suppress the D-V pattering defects caused by mutations in DNA repair enzymes. Interestingly, hus1 mutants are also characterized by a range of karyosome formation defects, much like mutants expressing defective DNA repair enzymes. These results suggested that during meiosis, hus1 is required for efficient activation of the meiotic checkpoint in response to persistent DSBs and is also essential for the organization of the oocyte DNA, a function that may be independent of the meiotic checkpoint (Peretz, 2009)
This study further analyzes the role of hus1 during meiosis; hus1 was found to be required for the efficient repair of DSBs during homologous recombination (HR) in meiosis. hus1 genetically interacts with brca2. It was also shown that non-repaired DSBs in the hus1 mutant lead to activation of a DmChk2 checkpoint. These results thus suggest that hus1 plays a role in the repair of meiotic DSBs (Peretz, 2009)
This study shows that the aberrant karyosome phenotype in the hus1 mutant is caused by defective homologous recombination (HR) repair. Histone γ-His2Av phosphorylation, a DSB marker, was dramatically increased in hus1 mutant flies and these persisted until later stages of oogenesis, as compared to wild-type flies. Additionally, blocking the formation of DSBs by using mei-W68 mutant flies suppressed the karyosome defect of hus1 mutant. The persistence of DSBs and karyosome defects in hus1 mutants resemble phenotypes found in flies with mutations in DNA repair enzymes of the spindle class genes. Taken together, these findings suggest that hus1 functions not only in activating the meiotic (pachytene) checkpoint but also in the repair of DSBs by HR during meiosis. Supportive of a role for hus1 in HR is the finding that reducing hus1expression in mouse cells by a siRNA approach decreases the efficiency of HR repair (Wang, 2006). Mammalian Rad9, a member of the Rad9-Hus1-Rad1 complex (9-1-1), interacts with Rad51, and inactivation of mammalian Rad9 results in decreased HR repair (Pandita, 2006). In yeast cells, it was shown that Rad17, the Rad9 homologue, and Rad24, the Rad17 homologue, are required for repair of DSBs during meiosis by facilitating proper assembly of the meiotic recombination complex containing Rad51, a protein which catalyzes DNA strand invasion. Therefore, it seems likely that the 9-1-1 complex as a whole could function during HR, this requires further examining (Peretz, 2009)
Interestingly, flies mutant for the recently identified Drosophila brca2 gene, are characterized by a highly penetrant karyosome defect, weakly ventralized eggs and persisting DBSs, implying a role for brca2 in homologous recombination repair. The abnormal D-V eggshell phenotype in mutants of DNA repair enzymes can be suppressed by mutations in brca2. This suggests that brca2 plays an additional role in transduction of the meiotic recombination checkpoint signal (Klovstad, 2008). It was reasoned that such a requirement for brca2 in activation of the checkpoint masks the strong eggshell ventralization phenotype normally characteristic of mutants of DNA repair enzymes (Klovstad, 2008). A similar rational could be applied to results with the hus1 mutant, where hus1 represents another protein with a dual function in both DNA repair and checkpoint activation during Drosophila meiosis. It is suggested that hus1 and brca2 thus represent a new class of proteins that serve a dual function in HR repair and in checkpoint activation during meiosis but whose mutant alleles do not show the full and/or strong repertoire of phenotypes of classic repair enzyme mutants (Klovstad, 2008). Interestingly, it was also reported that the Drosophila ATR homologue, mei-41, serves a dual function in DNA damage checkpoint and in facilitating the later stages of HR repair. mei-41 mutants also show a pattern of γ-His2Av staining in oocytes similar to that seen in DSB repair mutants, including delayed onset and persistence of foci into late pachytene (Joyce, 2009). The reduced or partial phenotypes displayed in mutants of these proteins (hus1, brca2 and mei-41), as compared to DNA repair enzyme mutants of the spindle class, may be indicative of a more regulatory natured role in repair, rather than a direct one. Thus, in DNA repair mutants a meiotic checkpoint is activated due to lack in repair of DSBs, while in mutants of regulatory genes (such as hus1, brca2 and mei-41) DSBs are also not repaired, however the checkpoint is not transduced properly leading to less pronounced phenotypes (Peretz, 2009)
It was also found that a mutation in the DmChk2 gene was able to suppress the karyosome and SC disassembly defects observed in hus1 mutant egg chambers, although DSBs persisted in these double mutants. This implies that in flies lacking hus1, DSBs are not repaired and this, in turn, leads to the activation of a DmChk2-dependent checkpoint. A mei-41 mutation was, however, unable to suppress these karyosome and SC defects. Similar results were reported for brca2 mutants, where the karyosome defects were attributed to activation of DmChk2 checkpoint but not of mei-41dependent one (Klovstad, 2008). Supporting evidence to the inability of mei-41 to suppress hus1 karyosome defects is the finding that mei-41 (Joyce, 2009) but not DmChk2 (this study) mutants show defects in processing DSBs during meiosis. The activation of a DmChk2-dependent checkpoint in hus1 mutants could be due to activation of DmChk2 by the other upstream checkpoint kinase, ATM. At this point, using the karyosome suppression assay, it was not possible to test whether ATM is the upstream activator, since atm mutants themselves present karyosome defects. It will be interesting to test whether atm, as hus1 and brca2, has a dual role in activation of the meiotic checkpoint and in HR repair (Peretz, 2009)
Since Brca2 co-immunoprecipitated Rad9, a member of the 9-1-1 complex, (Klovstad, 2008) and this study demonstrated a dual function for hus1 in meiosis, which was similar to that of brca2, it was decided to test whether hus1 and brca2 genetically interact. Defects in oocyte localization and determination, which were found to be characteristic of other DNA repair enzymes, were used as an indirect outcome of DSB repair in the oocyte. A mutation in brca2 was shown to strongly enhance the oocyte localization and determination defects found in the hus1 mutants. The finding that both hus1 and brca2 mutants show defects in DSB repair in the oocyte and that Brca2 physically interacts with Rad9, a part of the 9-1-1 complex (Klovstad, 2008), suggest that hus1 and brca2 may be a part of the same pathway of HR repair (Peretz, 2009)
However, the genetic interaction between hus1 and brca2 in HR repair in the germarium pro-nurse cells could be interpreted in a different manner. In wild-type, in region 2a of the germarium some of the pro-nurse cells contained DSBs as revealed by γ-His2Av staining. In later stages of meiosis, region 2b-3, the DSBs were restricted only to the oocyte, suggesting that there is a mechanism that ensures the restriction of DSBs to the oocyte and prevents these breaks in pro-nurse cells. It was found that in the double mutant flies for hus1 and brca2, but not in the single mutants, all of the germaria nurse cells showed γ-His2Av staining, suggesting that the nurse cells throughout the germarium have DSBs. These results point towards the role of hus1 and brca2 in DSB repair both in the pro-nurse cells and the oocyte. Such defects in DSB repair in the nurse cells and the oocyte could be the cause for the apoptosis of egg chambers in brca2;hus1 double mutant flies. The finding that the defects in DSB repair in the pro-nurse cells were detected only in the double brca2;hus1 mutants but not in the single ones, suggests that in this process hus1 and brca2 could act in parallel or in redundant pathways. Since it was shown that mei-41 mutants do cause a persistence of γ-His2Av foci in the oocyte but not in the nurse cells (as in the hus1 mutant), it will be interesting to study the complex interactions between mei-41, brca2 and hus1 in this process (Joyce, 2009). Altogether, these results lead to the identification of Hus1 as a protein with a dual role in activation of the meiotic checkpoint and in HR repair during meiosis (Peretz, 2009)
The human cancer susceptibility gene, BRCA2, functions in double-strand break repair by homologous recombination, and it appears to function via interaction of a repetitive region ("BRC repeats") with RAD-51. A putatively simpler homolog, Brca2, was identified in Drosophila recently and also affects mitotic and meiotic double-strand break repair. This study examined patterns of repeat variation both within D. pseudoobscura and among available Drosophila genomes sequences. Extensive variation was identified within and among closely related Drosophila species in BRC repeat number, to the extent that variation within this genus recapitulates the extent of variation found across the entire animal kingdom. Patterns of evolution were described across species by documenting recent repeat expansions (sometimes in tandem arrays) and homogenizations within available genome sequences. Overall, patterns and modes of evolution were documented in a new model system of a gene which is important to human health (Bennett, 2009).
Search PubMed for articles about Drosophila Brca2
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 ID: 17327271
Bennett, S. M. and Noor, M. A. (2009). Molecular evolution of a Drosophila homolog of human BRCA2. Genetica 137(2): 213-9. PubMed ID: 19554456
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 ID: 17822964
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 ID: 17363252
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 ID: 17515903
Delacroix, S., et al. (2007). The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev. 21: 14721477. PubMed ID: 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 ID: 17515904
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 ID: 17432935
Joyce, E. F. and McKim, K. S. (2009). Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation. Genetics 181: 39-51. PubMed ID: 18957704
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 ID: 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 ID: 11756561
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 ID: 12140769
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 ID: 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 ID: 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 ID: 9171368
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 ID: 15798199
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 ID: 10357855
Patel, K. J., et al. (1998). Involvement of Brca2 in DNA repair. Mol. Cell 1: 347357. PubMed ID: 9660919
Pandita, R. K. et al. (2006). Mammalian rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair. Mol. Cell. Biol. 26: 1850-1864. PubMed ID: 16479004
Peretz, G., Arie, L. G., Bakhrat, A. and Abdu, U. (2009). The Drosophila hus1 gene is required for homologous recombination repair during meiosis. Mech. Dev. 126(8-9): 677-86. PubMed ID: 19501158
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 ID: 16299390
Sekelsky, J. J., Brodsky, M. H. and Burtis, K. C. (2000). DNA repair in Drosophila: insights from the Drosophila genome sequence. J. Cell Biol. 150: F3136. PubMed ID: 10908583
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 ID: 15485900
Venkitaraman, A. R. (2002). Cancer Susceptibility and the Functions of BRCA1 and BRCA2. Cell 108: 171-182. PubMed ID: 11832208
Wang, X., Hu, B., Weiss, R. S. and Wang, Y. (2006). The effect of Hus1 on ionizing radiation sensitivity is associated with homologous recombination repair but is independent of non homologous end-joining. Oncogene 25: 1980-1983. PubMed ID: 16278671
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 ID: 15703751
Yu, V. P., et al. (2000). Gross chromosomal rearrangements and genetic exchange between nonhomologous chromosomes following BRCA2 inactivation. Genes Dev. 14: 14001406. PubMed ID: 10837032
date revised: 10 April 2010
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