Repair of Mismatched Templates during Rad51-dependent Break-Induced Replication

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3’ end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. When every 10 th base is mismatched between donor and recipient, BIR is 44% efficient compared to fully homologous sequences; but BIR still occurs about 10% when every 6 th base is mismatched. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, but where they are clustered with spacings of every 6 th bp. These different arrangements of uneven mismatch distribution were in general less efficient in recombination as templates with evenly distributed mismatches. A donor with all 10 mismatches clustered every 6 th base at the 3’ invading end of the DSB was not impaired compared to the case where mismatches were clustered at the 5’ end. These data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but sequence-specific factors are also important. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3’ end. Mismatch incorporation depends on the 3’ → 5’ proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins. Surprisingly, the probability of a mismatch 27 nt from the 3’ end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6 th base or fully homologous. These data suggest that DNA polymerase δ “chews back” the 3’ end of the invading strand without any mismatch-dependent cues from the strand invasion structure. Author Summary DNA double-strand breaks (DSBs) are the most lethal forms of DNA damage and inaccurate repair of these breaks presents a serious threat to genomic integrity and cell viability. Break-induced replication (BIR) is a homologous recombination pathway that results in a nonreciprocal translocation of chromosome ends. We used budding yeast Saccharomyces cerevisiae to investigate Rad51-mediated BIR, where the invading 3’ end of the DSB and a donor template share 108 bp of homology. We examined the tolerance of differently distributed mismatches on a homologous donor template and found that BIR efficiency was the same whether the mismatches were clustered at the 3’ invading end or at the 5’ end. We confirmed that mismatches are incorporated into the BIR product in a strongly polar fashion as far as about 40 nucleotides from the 3’ end. We conclude that the proofreading activity of DNA polymerase δ “chews back” the 3’ end of the invading strand even when the sequences removed have no mismatches for the first 26 nucleotides. These observations enrich our understanding of the details of Rad51-mediated strand invasion and provide insight into the mechanism of the 3’ to 5’ proofreading activity of DNA polymerase during homologous recombination.