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

Abstract

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 6th 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 6th 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 6th 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 6th 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.

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

“…These events still apparently do not involve Msh2-dependent mismatch correction (Fig. 5, and previous results (65, 72).…”

“…Almost all mismatch assimilation is lost in a pol3-01 proofreading-defective mutant. In fact, Pol3-dependent 3’ to 5’ resection could be demonstrated even if the first 26 nt of the 3’ invading end was identical to the donor (72).…”

“…In the haploid BIR assay, once heteroduplex DNA is formed by strand invasion, mismatches are assimilated into the BIR product in a strongly polar fashion (65). Mismatches close to the 3’ end of the invading strand are almost always replaced by the donor template sequence, with the likelihood of incorporating a mismatch declining rapidly until about 40 bp from the 3’ end (65, 72). The assimilation of the template sequence almost completely depends on the 3’ to 5’ proofreading exonuclease activity of DNA polymerase δ, which chews back the 3’ end of the invading strand before initiating new DNA synthesis (65, 72)(Fig.…”

“…Mismatch assimilation in dmc1 Δ hed1 Δ meiotic cells was not affected by deleting MSH2 , and heterologies were still incorporated as far as 45 bp from the 3’ end of the DSB, as in mitotic haploids and diploids (Fig. 5C) (72). However, in Dmc1-driven (wild type) meiotic cells there was less assimilation of mismatches near the 3’ invading end and in a small fraction of cases, the extent of donor sequence incorporation was extended over nearly the length of homology (Fig.…”

Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing inSaccharomyces cerevisisae

“…Possibly the cleaved end would be perceived in the way that a nicked strand is designated to be the corrected strand during DNA replication [33]. The presence of a nearby 3’ end can also employ the 3’ to 5’ exonuclease activity of DNA polymerase δ to chew back as far as 25 nt before then polymerizing new DNA (Figure 7C), similar to strongly biased repair of an invading strand during homologous recombination [14, 34, 35]. The processing of the 3’ end in this way would suggest that some of the gradient of repair that we observed is attributable to this alternative process (Figure 7).…”