Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae.

Abstract

We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828 and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T-*A base change at position Zll of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MATa switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z1lT strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Zl1A mutation reduced the efficiency of double-strand break formation compared with the Z1lT variant; the reduction was more severe in MATa than in MATa. In MATa, the Zl1A mutation also creates a matel (sterile) mutation that distinguishes switches of MATa-stk to either MATa or matkl-stk. Pedigree analysis of cells induced to switch in G, showed that MATa-stk switched frequently (23% of the time) to produce one matod-stk and one MATa progeny. This postswitching segregation suggests that Zil was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of matal -stkIMATa sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.Mating-type switching in the yeast Saccharomyces cerevisiae involves replacement of a or a DNA sequences at the MAT locus by opposite mating-type sequences derived from one of two silent donor loci, HMRa and HMLax (Fig. 1).cis-acting mutations that reduce or abolish MAT switching have been obtained (9,33,38,41). These mutations prevent efficient cleavage of the MAT locus by HO endonuclease, which initiates replacement of Ya or Ya by a gene conversion mechanism. Two types of mutations have been described. MATa-inc and MATa-inc (inconvertible) mutations are lesions within the HO recognition-cleavage site that are lost (healed) during rare switches to the opposite mating type because the double-strand break repair mechanism invariably replaces the region including these base pair changes. A second class of "stuck" mutations was described by Haber et al. (9). These mutants exhibited reduced switching from MATa to MATa but differed from inc mutants in that they were not healed in switching from MATa-stk to MATa (which switched normally) and back to MATa-stk (9). The MATa-stk mutations appeared to reduce the efficiency of HO endonuclease cleavage because HO MATa-stk strains carrying the rad52-1 mutation did not die, while wild-type MATa strains died when HO endonuclease-induced double-strand breaks were unable to be repaired.Another M...

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…They further showed that there was no reciprocal transfer of markers from MAT to the donor. This observation was supported by studies of the mismatch repair of a singlebp mutation only 8 bp from the 39 of the HO cut, in the Z region (Ray et al 1991). In the absence of mismatch repair, this mutation was most often retained during switching (thus confirming physical studies showing that there was almost no 39 to 59 removal of the 39-ended tail).…”

“…A site with only 21 bp results in inefficient single-strand nicking that, by replication, can be converted to a DSB (Cortes-Ledesma and Aguilera 2006). Single-base-pair MAT-inc (inconvertible) or MAT-stk (stuck) substitutions in the recognition site abolish or greatly reduce switching 8 (Weiffenbach and Haber 1981;Ray et al 1991). HO cutting generates 4 bp, 39-overhanging ends, both of which are accessible to exonucleases in vitro (Kostriken et al 1983).…”

Cell Cycle and Genetic Requirements of Two Pathways of Nonhomologous End-Joining Repair of Double-Strand Breaks in Saccharomyces cerevisiae

“…How such deletions might be formed is still a matter of conjecture. A large body of data from S. cerevisiae suggests that DSBs are degraded by 5Ј-to-3Ј exonucleases to create long stable single-stranded 3Ј ended tails (44,57,58,63). When the DSB is flanked by a substantial region of homology, the break can be repaired by SSA (11,31,57), which in S. cerevisiae requires the RAD1 and RAD10 genes to excise the nonhomologous tails present after annealing (10,20).…”

Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1

“…This ratio may reflect a repair bias intrinsic to the C͞A mismatch in this context, or it could reflect the influence of nearby heterologies. Alternatively, it could be caused by a directed bias in mismatch correction in much the same way as mismatch repair is biased during strand invasion during mitotic DSB-induced gene conversion (41,42). No apparent preference for the A or F allele was observed in strains defective in mismatch repair, suggesting that a residual repair pathway is unlikely to possess such a bias.…”