Distinct Regulation of Mlh1p Heterodimers in Meiosis and Mitosis in Saccharomyces cerevisiae

Abstract

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.

Genetic Analysis ofmlh3Mutations Reveals Interactions Between Crossover Promoting Factors During Meiosis in Baker’s Yeast

“…mlh3-K83A strains showed a wild-type phenotype (Table 3). Our results for the mlh3-N35A and mlh3-G97A mutations were similar to those obtained by Cotton et al (2010). However, for mlh3-E31A , which is thought to disrupt ATP hydrolysis by the Mlh3 subunit, we observed a null MMR phenotype; Cotton et al (2010) observed a close to wild-type phenotype for this mutant.…”

“…mlh1-E31A and mlh1-K84A , however, conferred MMR phenotypes that were different from their equivalent mlh3 mutations, with mlh1-E31A strains appearing more proficient in MMR and mlh1-K84A strains less proficient [Tables 3 and 4 (Tran and Liskay 2000; Hoffman et al 2003; Wanat et al 2007; Argueso et al 2003)]. Thus our work, in conjunction with previous studies, reinforces the hypothesis that the subunits of MLH complexes provide unique contributions to MMR (Tran and Liskay 2000; Hall et al 2002; Argueso et al 2003; Hoffman et al 2003; Wanat et al 2007; Nishant et al 2008; Cotton et al 2010). …”

“…Thus, our analyses are consistent with ATP hydrolysis by Mlh3 being important for its meiotic and MMR functions. We do not have a clear explanation for why our data differ from Cotton et al (2010). However, one possibility is that the SK1 strain background is more sensitized to defects in MLH3 compared with the Y55 background studied by Cotton et al (2010).…”

“…Like Cotton et al (2010), we found that the predicted ATP binding mutation mlh3-N35A conferred a null phenotype in the meiotic assays. However, in contrast to a nearly wild-type phenotype previously seen for mlh3-E31A in both MMR and meiotic assays (Cotton et al 2010), we found that mlh3-E31A mutants displayed, compared with the wild-type, defects in meiosis (Table 4; 67 cM map distance, 89% spore viability, P < 0.001) and MMR (null phenotype, Table 3). Thus, our analyses are consistent with ATP hydrolysis by Mlh3 being important for its meiotic and MMR functions.…”

“…We tested the effect of these mutations in a MMR repair assay that measures reversion of the lys2 :: InsE-A 14 allele (Tran et al 1997) and in meiotic assays that measure spore viability and crossing over in four intervals on chromosome XV in EAY1108/1112 SK1 congenic strains [Figure 2 (Argueso et al 2004)]. Three of the eight mlh3 mutations also were analyzed by Cotton et al (2010), using similar assays. In the lys2 :: InsE-A 14 reversion assay, mlh3Δ strains display a roughly 6-fold increase in mutation rate compared with wild-type (Harfe et al 2000; Nishant et al 2008; this study).…”