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

Abstract

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.3-kb of phage lambda DNA in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3′-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3 and Slx4. A natural 3% divergence between repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutSa (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutLa) are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the identical repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain, where the F variant carries six single-base mutations and one +1(T) insertion within a run of 10 Ts showed a gradient of correction favoring the sequence opposite the 3′ end of the annealed strand. Mismatches (MMs) located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, using either 3′ nonhomologous tail removal or 3′ to 5′ "proofreading" resection by DNA polymerase d followed by synthesis to fill in the gap. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat, with a bias in favor of the "left" copy. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, while the deletion of MSH6 resulted in unrepaired MMs.

Functions of PMS2 and MLH1 important for regulation of divergent repeat-mediated deletions

“…Namely, the bottom strand / 3' end is 16 bp from the edge of the repeat, whereas the top strand 5' DSB has 287 bp of non-homologous sequence. It is unclear how the length of the non-homologous tail may affect polar resolution of sequence divergence, but notably in yeast a 3' non-homologous tail is important for MMR engagement in divergent recombination events [52,53]. In any case, these findings indicate that the MLH1-KQQ motif is not absolutely required for polar resolution of sequence divergence.…”