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
). 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
). 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
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 (). 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
). 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.