Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae

We have studied deletion formation by single-strand annealing (SSA) between naturally divergent 200 bp repeats in budding yeast. SSA was initiated by creating a site-specific double-strand break (DSB) in the region between the two repeats, either using HO endonuclease or guide RNA-directed Cas9. In this process, the ends are resected 5’ to 3’, leaving long 3’-ended single-stranded sequences that can anneal. After annealing any 3’-ended nonhomologous tails are clipped off by the Rad1-Rad10 endonuclease. The presence of nonhomologous tails triggers a Msh2-Msh3-dependent and Sgs1-dependent heteroduplex rejection when there are 7 heterologies within the 200-bp repeats being annealed, whereas a “tailless” construct is not rejected.  Mismatch repair within the heteroduplex is Msh2-Msh6-dependent; however, a msh2 deletion proved have a spectrum of mismatch repair different from a msh3 msh6 double mutant, implying that Msh2 may have some additional role when the intermediates have nonhomologous 3’ tails. “Tailed” repeats yielded a gradient of repair that favored sequences in the “left” repeat, but “Tailless” SSA yielded a uniform correction of all 7 heterologies in the 200-bp region. A cluster of repeats near the left end of the sequence provokes correction of these markers in a Msh6-independent fashion; this correction is not attributable to 3’ to 5’ exonuclease activity of DNA polymerase d and may reflect instances where part of the homologous sequence itself is clipped off by the Ra1-Rad10 endonuclease. The presence or absence of nonhomologous tails also affected the requirement for the Rad52 strand annealing protein. Both in the chromosomal SSA assay and in a plasmid assay, Rad52 was largely dispensable when there were no nonhomologous tails after strand annealing.