Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can he separated. In this report we show that in Saccharomyces cerevisiae mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh 1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh 1 alleles displayed wild-type or nearly, wild-type mutation rates at 26degrees. At 35degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-1296S strains was partially suppressed at 35degrees by EXO1 overexpression. The mlh1-F228S and 4296S mutations conferred a separation-of-function phenotype in meiosis; both mlh 1-F228S and -1296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic Studies Suggest that the Conditional mlh1 mutations can be used to separate the MMR and meiotic crossing over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.