XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids

Abstract

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.

ATM- and ATR-Mediated Phosphorylation of XRCC3 Regulates DNA Double-Strand Break-Induced Checkpoint Activation and Repair

“…S6E in the supplemental material) (17). Consistent with our previous study (16), XRCC3-depleted cells exhibited an ϳ10-fold reduction in the I-SceI-induced GFP-positive cells ( Fig. 5F; see also Fig.…”

“…These paralogs act in the BRCA1/2 pathway to control HR (14, 15). The RAD51 paralogs differentially regulate both shortand long-tract gene conversions and have been implicated in the late stages of HR for resolving recombination intermediates (16,17). In addition to HR, RAD51 paralogs have been implicated in chromosome segregation and in the prevention of aberrant mitosis and aneuploidy (18,19).…”

Rad51 Paralog Complexes BCDX2 and CX3 Act at Different Stages in the BRCA1-BRCA2-Dependent Homologous Recombination Pathway

“…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).…”

“…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 (Ͼ1-kb) compared to short-tract (Ͻ1-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).…”

Noncanonical views of homology-directed DNA repair

“…Because LTGC in mammals also results in the formation of long recombination products, it has been suggested to be analogous to BIR in yeast (Willis et al 2015). Interestingly, depletion of BRCA1 or the Rad51 paralog XRCC3 increased the tract length of GC products (Brenneman et al 2002;Nagaraju et al 2009;Chandramouly et al 2013). The absolute frequency of such LTGC products after site-specific replication stalling was found to be enhanced after BRCA1 and BRCA2 knockdown in mammalian cells, suggesting that the loss of canonical HR pathways due to BRCA deficiency may enhance mutagenic recombination events at stalled replication forks (Willis et al 2014).…”