Analysis of the proteins involved in the in vivo repair of base-base mismatches and four-base loops formed during meiotic recombination in the yeast Saccharomyces cerevisiae

Abstract

DNA mismatches are generated when heteroduplexes formed during recombination involve DNA strands that are not completely complementary. We used tetrad analysis in Saccharomyces cerevisiae to examine the meiotic repair of a base-base mismatch and a four-base loop in a wild-type strain and in strains with mutations in genes implicated in DNA mismatch repair. Efficient repair of the base-base mismatch required Msh2p, Msh6p, Mlh1p, and Pms1p, but not Msh3p, Msh4p, Msh5p, Mlh2p, Mlh3p, Exo1p, Rad1p, Rad27p, or the DNA proofreading exonuclease of DNA polymerase delta. Efficient repair of the four-base loop required Msh2p, Msh3p, Mlh1p, and Pms1p, but not Msh4p, Msh5p, Msh6p, Mlh2p, Mlh3p, Exo1p, Rad1p, Rad27p, or the proofreading exonuclease of DNA polymerase delta. We find evidence that a novel Mlh1p-independent complex competes with an Mlhp-dependent complex for the repair of a four-base loop; repair of the four-base loop was affected by loss of the Mlh3p, and the repair defect of the mlh1 and pms1 strains was significantly smaller than that observed in the msh2 strain. We also found that the frequency and position of local double-strand DNA breaks affect the ratio of mismatch repair events that lead to gene conversion vs. restoration of Mendelian segregation.

Figure 1.—

Comparison of segregation patterns in strains with varying levels of DSB hotspot activity. The observed percentages of total aberrant segregation, gene conversion, and postmeiotic segregation events are indicated as Total Ab., GC, and PMS, respectively. As described in the results, we calculated restoration events (Rest.) by comparing the frequencies of aberrant segregation in wild-type and msh2 strains. (A) Strains with wild-type DSB frequency at the HIS4 hotspot. (B) Strains with wild-type DSB frequencies at the ARG4 and DED81 hotspots. (C) Strains homozygous for an insertion of telomeric sequence upstream of ARG4 (arg4-tel) that increases the DSB frequency at the ARG4 hotspot and decreases the DSB frequency at the DED81 hotspot. (D) Strains homozygous for the bas1 deletion, a deletion that eliminates the DSB located immediately upstream of HIS4. (E) Strains homozygous for the his4-51 mutation. This mutation eliminates the Rap1p-binding site upstream of HIS4 and eliminates the HIS4-associated DSB.

Figure 2.—

Comparison of the MutL and MutS homologs involved in the MMR spellchecker function (repair of errors introduced by DNA polymerase) and the MMR of mismatches formed during meiotic recombination. The relative contributions of each complex to the repair of base–base mismatches and four-base loop substrates are indicated by the arrow size. Msh2p/Msh6p/Mlh1p/Pms1p is involved in MMR of base–base mismatches in both contexts. Msh2p/Msh3p/Mlh1p/Pms1p initiates the majority of repair of four-base loops for the spellchecker function, with Msh2p/Msh3p/Mlh1p/Mlh2p and Msh2p/Msh3p/Mlh1p/Mlh3p making very small contributions (Wang et al. 1999; Harfe and Jinks-Robertson 2000). The meiotic repair of four-base loops is initiated primarily by Msh2p/Msh3p/Mlh1p/Pms1p, but an Mlh1-independent, Mlh3p-dependent complex (possibly Msh2p/Msh3p/Mlh3p/Mlh3p) is responsible for about one-third of the repair events.

Figure 3.—

Nick-directed mismatch repair. (A) Mismatch repair directed by the nick that initiates DSB formation (Detloff et al. 1992; Porter et al. 1993) or by the nick that is involved in resolving the recombination intermediate (Foss et al. 1999; “late repair”). Recombination is initiated by a DSB (indicated by large arrowhead) on the wild-type (blue) chromosome, and the broken ends are resected (step 1). After strand invasion, DNA synthesis extends the D-loop, resulting in a mismatch within the heteroduplex DNA (step 2). Steps 3–5 illustrate repair that is directed by the nick remaining at the initiation site (the I nick). In step 3, DNA from the I nick through the mismatch is excised. DNA synthesis subsequently fills in the gap, duplicating the mutant information (step 4). I-nick-directed repair results in a gene conversion event (step 5). Steps 3′–6′ depict repair that is directed by nicks involved in resolving the Holliday junctions (the R nicks). In steps 3′ and 4′, the DNA strands that have been involved in the exchange are nicked (shown by small arrowheads). In step 5′, DNA is excised from the R nick through the mismatch. DNA synthesis then replicates the wild-type information (step 6′), resulting in a restoration event. (B) Repair directed by DSBs initiated in a neighboring gene. Southern analysis demonstrates that there are two preferred sites for DSB formation near ARG4, one immediately upstream of ARG4 and a second upstream of the neighboring DED81 gene (Nicolas et al. 1989; Fan et al. 1995). In wild-type strains, more DSBs are generated at DED81 than at ARG4, as indicated by the size of the arrows. We suggest that the majority of restoration-type repair of the arg4-17 mismatch are the result of the processing of an R nick of a heteroduplex initiated by the DED81 DSB. A second source of restoration events is excision from an R nick of a heteroduplex initiated by the ARG4 DSB, as shown in A, steps 3′–6′.