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The Rad51 paralogs are required for homologous recombination (HR) and the maintenance of genomic stability. The molecular mechanisms by which the five vertebrate Rad51 paralogs regulate HR and genomic integrity remain unclear. The Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 and CX3 complexes act at different stages of the HR pathway. In response to DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment but upstream of Rad51 recruitment. In contrast, the CX3 complex acts downstream of Rad51 recruitment but still has a marked impact on the measured frequency of homologous recombination. Both complexes are epistatic with BRCA2 and synthetically lethal with Rad52. We conclude that human Rad51 paralogs facilitate BRCA2-Rad51-dependent homologous recombination at different stages in the pathway and function independently of Rad52.
Homologous recombination (HR) is an error-free mechanism of repairing double-strand breaks (DSBs) that can occur during the mitotic cell cycle that arise from endogenous DNA damage or exogenously by exposure to radiation and other DNA-damaging agents (1). Abnormalities of HR are associated with a number of genetic diseases, including ataxia-telangiectasia, Nijmegen break syndrome, Fanconi anemia, and Bloom's syndrome (2). HR abrogation in these diseases often leads to cancer susceptibility, which highlights the critical role of HR in maintaining genomic integrity (2). A key step in HR is the polymerization of Rad51 at 3′ single-stranded DNA (ssDNA) that is exposed after DSB processing by the Mre11-Rad50-Nbs1 (MRN) complex, CtIP, Exo1, and DNA2 nucleases (3). Rad51 nucleoprotein filament formation is required for subsequent homology search and strand invasion (4). In eukaryotic cells, BRCA2 is the primary mediator that facilitates Rad51 filament formation (5). Recent results from our laboratory now demonstrate that in the absence of BRCA2, Rad52 provides a secondary pathway to promote Rad51-mediated HR (6). Depletion of both BRCA2 and Rad52 is synthetically lethal, mirroring the phenotype of Rad51 inactivation. The recombinase activities of Rad51 in both pathways are well defined; however, the molecular mechanism by which the five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) regulate HR and genomic integrity remains unclear.
Multiple approaches have demonstrated that Rad51 paralogs are required for HR repair. Hamster cells or chicken DT40 B-lymphocyte cells defective in any of the Rad51 paralogs are sensitive to DNA cross-linking agents and to ionizing radiation (IR) (7–11). These mutant cells also display chromosomal aberrations, reduced frequencies of HR-mediated gene targeting and DSB repair, and reduced frequencies of sister chromatid exchanges (7–11). Rad51 paralogs may play an early role in the HR pathway, as they have been shown to bind single-stranded DNA (ssDNA) and single-stranded regions on duplex DNA (12). Importantly, Rad51 paralog deficiency significantly reduces Rad51 recruitment to damage foci in response to ionizing radiation (IR), suggesting that Rad51 paralogs facilitate Rad51 mediator activities (10, 13). The Rad51 paralogs are also linked to activities in later stages in the HR pathway post-Rad51-mediated strand invasion. The Rad51 paralogs appear to regulate gene conversion tract length (14, 15) and have been linked to Holliday junction (HJ) resolvase activity (16, 17). In addition, electron microscopy images show Rad51 paralogs binding to Y-shaped replication-like intermediates and synthetic HJs, supporting the idea of a role for Rad51 paralogs in repair during DNA replication and in resolution of HR intermediary structures (18). In mice, disruption of any Rad51 paralog genes leads to early embryonic lethality with accumulation of unrepaired DNA damage (19–21). These results demonstrate that Rad51 paralogs are essential for preserving genomic integrity through their activities in HR repair both in early stages of development and in normal mitotic cells.
The Rad51 paralogs have 20% to 30% homology with Rad51 and perhaps arose by gene duplication and evolved new functions. In Saccharomyces cerevisiae, the Rad51 paralogs Rad55 and Rad57 form a heterodimeric complex. Biochemically, vertebrate Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3) (12). Genetic studies in DT40 cells confirmed that the BCDX2 and CX3 complexes observed biochemically are also functionally distinct (22). The CX3 complex was shown to catalyze strand exchange in vitro (23). Similarly, the BC and DX2 subcomplexes of the BCDX2 complex have also been reported and shown to have in vitro Rad51 mediator activity (24) and strand exchange activity (25), respectively. However, the specific activities of the Rad51 paralog complexes and subcomplexes have not been defined in vivo.
To better understand the role of the Rad51 paralogs, we sought to determine where they are required in the known HR pathway. Depletion of components in the BCDX2 complex or the CX3 complex in human cells did not affect BRCA1 or BRCA2 recruitment to damage foci. In contrast, Rad51D depletion decreased Rad51 recruitment to damage sites whereas depletion of XRCC3 did not. Furthermore, XRCC3−/− human colorectal cells did not show a defect in Rad51 focus formation after IR exposure. Since Rad51D depletion represents a loss in the BCDX2 complex and XRCC3 depletion or deletion represents a loss in the CX3 complex, the data suggest that the BCDX2 and CX3 complexes act at different stages in the HR pathway. Double depletion of components in the BCDX2 complex (Rad51D or XRCC2) with components in the CX3 complex (Rad51C or XRCC3) did not lead to additive loss of Rad51 focus formation, further demonstrating that the BCDX2 complex, and not the CX3 complex, is responsible for Rad51 recruitment or stabilization at damage sites. Since BRCA2 and Rad52 provide two independent pathways for Rad51-mediated HR in mammalian cells (6), we also demonstrate that in human cells, both complexes are epistatic with the BRCA2 pathway of HR.
The MCF7 human breast carcinoma cell line and EUFA423 human TERT (hTERT)-transformed fibroblasts with compound heterozygous mutations in BRCA2 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine growth serum (BGS), 20 mM HEPES, 100 μg/ml streptomycin, and 100 units/ml penicillin (Sigma). U2OS cells were grown in DMEM supplemented with 10% BGS and 2 mM l-glutamine. HCT116 parental cells, HCT116 XRCC3−/− cells, and HCT116 XRCC3−/− cells stably complemented with XRCC3 were grown in McCoy's 5a media (Invitrogen) supplemented with 10% BGS, 2 mM l-glutamine, and 20 mM HEPES. The Qiagen small interfering RNA (siRNA) oligonucleotides used to transiently deplete the Rad51 paralogs were as follows: nontarget (NT) control (catalog no. 1027281), Rad51B_1 (SI00045080), Rad51B_6 (SI03074232), Rad51C_6 (SI02663689), Rad51C_7 (SI02757608), Rad51D_2 (SI00045101), Rad51D_1 (SI00045094), XRCC2_6 (SI02665110), XRCC2_2 (SI00077091), XRCC3_1 (SI00077112), XRCC3_2 (SI00077119), and XRCC3_3 (SI00077126). BRCA2 was depleted using ON-TARGETplus Smart pool siRNA (catalog no. L-003462-00-0005; Dharmacon), and Rad52 was depleted using Silencer predesigned siRNA (catalog no. 142431; Ambion). BRCA2 was transiently complemented in EUFA423 cells using a pcDNA3B/HA-BRCA2 plasmid. siRNA and pcDNA3B/HA-BRCA2 transfections were carried out using Amaxa Nucleofector and the manufacturer's recommendations. pCMV-I-SceI-3xNLS or pCMV 3xNLS empty vector was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
Whole-cell extracts or nuclear fractions were used for Western blot analysis. Whole-cell extracts were prepared by lysing cells in radioimmunoprecipitation buffer (150 mM NACl, 50 mM Tris [pH 8.0], 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1.0% Nonidet P-40, 2.0 mM phenylmethylsulfonyl fluoride, 1.0 mM Na3VO4, Halt protease inhibitor cocktail [Pierce]). Nuclear fractions were isolated using a nuclear extraction kit (Active Motif) according to the manufacturer's instructions. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay reagent system (Bio-Rad). Protein (50 μg) was loaded into 10% Bis-Tris or 3% to 8% Tris acetate (for BRCA2 detection) precast gels (Invitrogen), subjected to SDS-PAGE, transferred onto a polyvinylidene difluoride Immobilon P membrane (Millipore), and blocked with 5% milk–TBST (50 mM Tris [pH 7.5], 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. Immunodetection was performed using the following antibodies: anti-Rad51C (NB100-177), anti-Rad51D (NB100-166), anti-XRCC2 (NB120-2367), and anti-XRCC3 (NB100-180) antibody from Novas Biologicals; anti-Rad52 (sc-8350) and anti-Rad51B (sc-53430) antibody from Santa Cruz Biotechnologies; anti-BRCA2 (CA1033) antibody from Calbiochem; antiactin (4967) antibody from Cell Signaling; anti-histone deacetylase 1 (06720) antibody from Upstate Biotechnologies; or anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (9484) antibody from Abcam. Goat anti-mouse IG and goat anti-rabbit IgG conjugated to horseradish peroxidase were used for secondary antibodies. Bands were detected using ECL chemiluminescence detection methods (PerkinElmer) and exposure to X-ray film (Molecular Technologies).
The pDR-GFP plasmid was stably integrated into MCF7, EUFA423, and U2OS cell lines as previously described (9). A Nucleofector (Amaxa) cell electroporator was used to transfect 0.5 × 106 cells with either 2 μg of siRNA for single depletion or 4 μg of siRNA for double depletions. Cells were then split into two wells of a 6-well plate. After 24 h, cells were transfected with 2 μg of pCMV-ISceI-3xNLS or pCMV-3xNLS empty vector and 1 μg siRNA for single depletions and 2 μg siRNA for double depletions using Lipofectamine 2000 (Invitrogen). After 72 h, cells were harvested and percentages of green fluorescent protein (GFP)-positive cells determined by flow cytometry (FACSCalibur; Becton, Dickinson) as previously described (26). For each siRNA condition, the percentage of GFP-positive cells in the empty vector control was subtracted from the percentage of pCMV-3xNLS-transfected cells.
A Nucleofector (Amaxa) cell electroporator was used to transfect 1 × 106 cells with the indicated siRNA oligonucleotides. Cells were grown on 10-cm-diameter dishes for 24 h and then trypsinized and seeded onto an 8-chamber tissue culture slide (Millipore) at 4 × 104 cells per well. These cells were incubated overnight and then treated with 10 Gy IR using a Mark1 generator. HCT116 parental cells, HCT116 XRCC3−/− cells, and HCT116 XRCC3−/− cells stably complemented with XRCC3 were plated at 8 × 104 cells per well and incubated for 48 h before treatment with 10 Gy IR. At the times indicated, cells were fixed with a 10% paraformaldehyde solution at room temperature for 15 min followed by a 30-min block and permeabilization in phosphate-buffered saline (PBS) containing 10% BGS and 0.5% Triton X-100. Cells were stained using Santa Cruz Biotechnologies anti-BRCA1 (sc-6954), anti-BRCA2 (sc-21230), and anti-Rad51 (sc-8349) antibodies in PBS containing 10% BGS and 0.5% Triton X-100. Secondary antibodies Alexa Fluor 596-labeled goat anti-mouse IgG, Alexa Fluor 488–goat anti-rabbit IgG, and Alexa Fluor 488–chicken anti-goat IgG (Molecular Probes) were used at a 1:500 dilution in PBS containing 10% BGS and 0.5% Triton X-100. After the primary and secondary antibodies were applied, cells were washed 3 times with PBS containing 0.5% Triton X-100. Images were obtained using a Zeiss LSM 510 Meta scanning confocal microscope. Maximum-intensity images were generated in the Zen program to display foci in all sections and were analyzed using ImageJ software. At least 100 nuclei were counted, with cells forming >5 foci scored as positive. The data shown represent averages of the results of at least 3 independent experiments, with error bars representing standard deviations (SD).
MCF7 cells (1 × 106) were Nucleofector-transfected with 2 μg of siRNA, plated on a 10-cm-diameter dish, and incubated for 24 h. Cells were seeded at 500, 200, 100, and 50 cells per well and grown for 14 days. Colonies were visualized by fixing with methanol and staining with crystal violet.
Samples with a normal distribution were analyzed by Student's t test. Differences between groups were considered significant at P < 0.05. Statistical analysis was performed using Graph-Pad Prism 5 (Graph-Pad Software).
To investigate Rad51 paralog activity in human cells, individual Rad51 paralogs were depleted in human breast carcinoma (MCF7) and human osteosarcoma (U2OS) cell lines using siRNA (Fig. 1a). Consistent with previous reports (27), depletion of Rad51 paralogs within one complex affected the stability of other members of the same complex. In both MCF7 and U2OS cells, Rad51B depletion led to decreases in levels of Rad51D and XRCC2 and Rad51C depletion led to a decrease in levels of XRCC3. Protein levels of Rad51D and XRCC2 were dependent on each other, as depletion of one led to a decrease in the other. Although Rad51C was in both complexes, depletion of Rad51C only slightly decreased or had no effect on levels of Rad51B, Rad51D, or XRCC2. These data suggest that siRNA depletion of Rad51C may be sufficient to affect the CX3 complex but not the BCDX2 complex. Rad51 expression was not affected by siRNA depletion of any Rad51 paralog (Fig. 1a; see also Fig. S4 in the supplemental material). As expected, depletion of any Rad51 paralog resulted in significant decreases in HR frequency measured by I-SceI-induced homology-mediated repair (Fig. 1b). To address the potential off-target effects of the presence of a single siRNA oligonucleotide, we tested at least two independent siRNAs for each Rad51 paralog (see Fig. S1 in the supplemental material).
Since previous studies suggested that the Rad51 paralogs act at both early (pre-Rad51 filament formation) and late (post-Rad51 filament formation) stages of the HR pathway, we tested the recruitment of BRCA1, BRCA2, and Rad51 to damage foci in cells treated with a nontarget control (NT), Rad51D, Rad51C, or XRCC3 siRNA. NT-treated MCF7 cells exposed to 10 Gy IR had an increase in BRCA1, BRCA2, and Rad51 focus formation (Fig. 2 and Fig. 3; see also Fig. S2 in the supplemental material). Depletion of Rad51D, Rad51C, or XRCC3 did not affect BRCA1 or BRCA2 recruitment to damage foci (Fig. 2; see also Fig. S2 in the supplemental material), suggesting that both Rad51 paralog complexes play a role downstream of BRCA1 or BRCA2 recruitment. In contrast, Rad51 focus formation was decreased in Rad51D-depleted cells but not XRCC3-depleted cells (Fig. 3). Rad51C depletion resulted in an intermediate decrease in Rad51 focus formation, consistent with the Western blot data in Fig. 1, which showed Rad51C depletion having a more pronounced effect on the CX3 complex than on the BCDX2 complex (Fig. 3). The kinetics of Rad51 focus formation showed that, at all time points tested, there were no differences in XRCC3-depleted cells (see Fig. S2 in the supplemental material). Therefore, a possible delay in Rad51 recruitment to damage sites in XRCC3-depleted cells was not the explanation. The result observed in MCF7 cells followed the same pattern as that seen in U2OS cells (see Fig. S2 and S3 in the supplemental material). Since siRNA depletion of XRCC3 does not completely eliminate XRCC3 expression, we tested Rad51 focus formation in XRCC3−/− human colorectal HCT116 cells with or without complementation with XRCC3 (Fig. 4). Loss of XRCC3 expression in the XRCC3−/− cells and the presence of XRCC3 in the stably complemented (XRCC3−/−+XRCC3) cells were confirmed by immunoblot analysis (Fig. 4a). Consistent with our siRNA results, we observed no difference in the levels of Rad51 focus formation at 1.5 and 5 h after treatment with 10 Gy IR in XRCC3-proficient compared to XRCC3-deficient cells (Fig. 4b and andc).c). To verify that the BCDX2 complex and the CX3 complex did not cooperate in Rad51 filament formation, we depleted both complexes and found no additive decrease in Rad51 focus formation after treatment with 10 Gy IR in both MCF7 and U2OS cells (Fig. 5; see also Fig. S5 in the supplemental material). Immunoblots showed that double depletions decreased levels of the appropriate Rad51 paralogs (Fig. 5a; see also Fig. S5a in the supplemental material). Rad51D depletion alone or in combination with Rad51C or XRCC3 resulted in a 40% decrease in Rad51 focus formation in both MCF7 and U2OS cells (Fig. 5; see also Fig. S5 in the supplemental material). Similarly, XRCC2 depletion alone or in combination with Rad51C or XRCC3 resulted in 30% and 40% decreases in Rad51 focus formation in U2OS and MCF7, respectively (Fig. 5; see also Fig. S5 in the supplemental material). Taken together, the results suggest that in human cells, the two paralog complexes act at different stages of the HR pathway; the BCDX2 complex acts upstream and the CX3 complex acts downstream of Rad51 recruitment to damage sites.
Since Rad51-mediated HR repair in human cells can occur through either the BRCA2- or Rad52-dependent pathway, we sought to determine whether the Rad51 paralogs were epistatic with BRCA2 or Rad52. Expression of the individual Rad51 paralogs was silenced in BRCA2 siRNA-depleted cells and analyzed for I-SceI-induced HR frequencies. Immunoblots to confirm double depletions are shown in Fig. S4 in the supplemental material. BRCA2 depletions led to 10- and 100-fold reductions in HR repair in MCF7 and U2OS cells, respectively (Fig. 6a and andb).b). Simultaneous Rad51 paralog depletions did not further reduce HR frequency in BRCA2-depleted cells, suggesting that the Rad51 paralogs are epistatic with BRCA2. In contrast, simultaneous depletion of Rad52 and BRCA2 resulted in a further decrease in HR, as previously reported (6). Additionally, double depletion of Rad51D and BRCA2 resulted in decreases in plating efficiency similar to those seen with single depletions, further supporting the idea of Rad51 paralog epistasis with BRCA2 (Fig. 6c). Rad51D siRNA depletion in BRCA2-deficient EUFA423 cells expressing a truncated BRCA2 protein also did not show a significant decrease in HR frequencies compared to NT siRNA-treated cells. Transient complementation of BRCA2 led to a 2-fold increase in HR, which slightly decreased with Rad51D siRNA (Fig. 6d).
Rad51 paralog epistasis with the BRCA2 pathway was further demonstrated by Rad51 paralogs being synthetically lethal with Rad52. Simultaneous depletion of any Rad51 paralog with Rad52 lead to a further decrease in I-SceI-induced HR (Fig. 7a). Immunoblots to confirm double depletions are shown in Fig. S4 in the supplemental material. Consistent with the HR data, Rad51 focus formation was further decreased in Rad51D- and Rad52-depleted cells compared to that seen with single depletions (Fig. 7b). Depletion of Rad52 in combination with Rad51D or XRCC3 led to a further decrease in plating efficiency, confirming the synthetically lethal relationship between the two Rad51 paralog complexes and Rad52 (Fig. 7c).
In this report, we demonstrate the Rad51 paralog complexes acting at different stages in the HR pathway: the BCDX2 complex acts upstream and the CX3 complex acts downstream of Rad51 recruitment to damage foci (see Fig. S6 in the supplemental material). Rad51 paralogs may act at early stages in HR by facilitating the assembly or stability of Rad51 nucleoprotein filament. Data to support this role include observations in Rad51 paralog-deficient hamster, chicken, and human cell lines lacking the ability to form Rad51 foci in response to IR. In addition, the yeast Rad51 paralogs, the heterodimeric Rad55/Rad57 complex, were shown to stabilize the Rad51 nucleoprotein filament by preventing disruption by the Srs2 antirecombinase (28). Rad55 is closest in homology to XRCC2, and Rad57 is closest in homology to Rad51D (29). Rad51D, which also forms a subcomplex with XRCC2, may therefore similarly stabilize Rad51 filament formation. Interestingly, the yeast and human Rad51 paralogs associate with components of the “suppress sgs1 hydroxyurea (HU) sensitivity” (Shu) complex, which also inhibits Rad51 filament disassembly by Srs2 (30, 31). In Saccharomyces cerevisiae, the Shu complex is comprised of four subunits, Csm2, Psy3, Shu1, and Shu2, which, when mutated, can suppress defects associated with sgs1 or top3 mutants (32–36). The components of the human Shu complex have yet to be identified and characterized. Interestingly, Shu1 and Psy3 are homologous with human XRCC2 and Rad51D, respectively, and Shu2 is homologous with human SWS1, which interacts with Rad51 and Rad51 paralogs through hSWS1-associated protein 1/C19orf39 (31). The human homolog of the antirecombinase Srs2 has yet to be defined; however, likely candidates include RECQL5 and PCNA-associated recombination inhibitor (PARI), which also negatively regulates HR by disrupting the Rad51 nucleoprotein filament (37, 38).
XRCC3 depletion significantly decreased HR activity but did not affect Rad51 focus formation in both the MCF7 and U2OS cell lines. Furthermore, XRCC3−/− human colorectal cells had no defect in Rad51 focus formation, in contrast to previous reports, which demonstrated Rad51 focus defects in these cells (39). Simultaneous depletion of both Rad51 paralog complexes did not lead to a further decrease in Rad51 focus formation, supporting the hypothesis that the CX3 complex does not contribute to Rad51 recruitment to damage sites. We therefore conclude that XRCC3 facilitates HR at later stages in the pathway that occur post-Rad51 recruitment to damage sites.
Although Rad51C is in both complexes, it displays an intermediate effect on Rad51 focus formation. Depletion of Rad51C by siRNA may be sufficient to remove the CX3 complex, a hypothesis which is supported by our immunoblot analysis, demonstrating greater depletion of XRCC3 than of components of the BCDX2 complex in Rad51C-depleted cells. Alternatively, the DX2 subcomplex may have a greater role than the BC subcomplex or the BCDX2 complex in Rad51 filament stabilization.
Increasing evidence now shows that the Rad51 paralogs act at later stages in the HR pathway. Kinetic data show that Rad51C foci appear rapidly and colocalize with Rad51 but persist long after Rad51 foci disappear (40). XRCC3 mutant irs1-SF cells have increased gene conversion tract lengths, increased frequencies of discontinuous tracts, and frequent local rearrangements (14), suggesting deficiencies in later stages of HR. Further support for the idea of a role of Rad51 paralogs in gene tract conversion length was also demonstrated using a reporter assay which showed a bias toward long-tract (>1-kb) compared to short-tract (<1-kb) sister chromatid recombination (15) in XRCC3 and XRCC2 mutant hamster cells. In addition, the CX3 complex, but not XRCC2, was shown to associate with HJ resolvase activity in cell fractionation experiments (16). The human HJ resolvases MUS81, GEN1, and SLX1/SLX4 complex have recently been identified (41). In light of these findings, the role of the CX3 complex in HJ resolution is unclear. Although it is unlikely that the CX3 complex is acting as a cooperating factor in resolvase activity, the idea of a role of stabilizing gene conversion tracts beyond the stage of Rad51 filament formation seems strongly supported by both our data and previous literature. Elucidation of the precise activity of CX3 would require development of in vivo approaches to study HR repair intermediates beyond Rad51 focus formation.
Finally, we suggest that both paralog complexes act in the BRCA2-dependent HR pathway (Fig. 6). Rad51 paralogs appear to be epistatic with BRCA2 and synthetically lethal or sick with respect to Rad52, which can independently mediate HR. BRCA2-depleted cells have a more profound defect in HR than any Rad51 paralog-depleted cells, which is likely due to BRCA2 having a direct role in loading Rad51. In contrast, the Rad51 paralogs may participate in stabilizing the Rad51 filament by inhibiting an antirecombinase, which may not reduce HR to the same extent. Although epistasis analysis in human cells using siRNA depletion approaches must be interpreted with caution, our results are consistent with data from genetic deletion mutants in DT40 chicken cells that demonstrate a synthetic lethal relationship between Rad52 and XRCC3 (42).
We have presented data investigating HR repair of two-ended double-strand breaks that were generated by ISceI-induced lesions or IR treatment. It should be interesting to investigate whether the results would extrapolate to one-ended double-strand breaks during impaired replication fork progression. We postulate that the Rad51 paralogs would function at least similarly, since they have hypersensitivity to fork replication-stalling agents such as DNA cross-linking agents and topoisomerase inhibitors. The Rad51 paralogs may in fact have a more pronounced role in the repair of replication-stalling lesions, since they have greater hypersensitivity to these agents than to IR.
The molecular mechanisms of the eukaryotic Rad51 paralogs have been difficult to define since their initial characterization almost two and half decades ago (7, 8). Our investigation has now shown the two Rad51 paralog complexes acting at different stages of the HR pathway. We also demonstrated that Rad51 paralogs are epistatic with BRCA2 and synthetically lethal with Rad52.
We thank Maria Jasin and Kiyoshi Miyagawa for providing us with HCT116 parental cells, HCT116 XRCC3−/− cells, and HCT116 XRCC3−/− cells stably complemented with XRCC3.
This work was supported by USPHS grant CA107640.
Published ahead of print 12 November 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00465-12.