Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

Abstract

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52 ؊/؊ mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

“…To verify that this method specifically measures SSA, we compared the effect of the knockdown of RAD52 (which is critical for SSA) and of BRCA2 (which is critical for HR). 23 We observed that BRCA2 knockdown specifically impaired HR, whereas RAD52 knockdown specifically impaired SSA (Fig. 3A).…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…Either BRCA1 or BRCA2 mutation reduces the efficiency of HR induced by a site-specific DSB [83][84][85][86][87]. Errorprone repair of the DSB by single strand annealing (SSA) is increased in BRCA2 mutants [87,88]-a pattern reminiscent of HR mutants in yeast and in other vertebrate cells. In contrast, SSA is partly dependent on BRCA1, suggesting that BRCA1 also participates in an earlier step in HR, perhaps working with the MRN complex to control processing of the DSB end [88,89].…”

“…Errorprone repair of the DSB by single strand annealing (SSA) is increased in BRCA2 mutants [87,88]-a pattern reminiscent of HR mutants in yeast and in other vertebrate cells. In contrast, SSA is partly dependent on BRCA1, suggesting that BRCA1 also participates in an earlier step in HR, perhaps working with the MRN complex to control processing of the DSB end [88,89]. These studies do not assay HR at stalled forks, since the induced DSB has no relation to replication arrest.…”

The C/A polymorphism in intron 11 of the XPC gene plays a crucial role in the modulation of an individual’s susceptibility to sporadic colorectal cancer

“…Most studies investigating the NBS1 Glu185Gln polymorphism have focused on assessing the risk of breast cancer [32]. There is only one publication on this NBS1 polymorphism and risk of colorectal cancer [33]. The XRCC3 protein interacts with Rad51-related proteins in the process of homologous recombination.…”