Systematic mutagenesis of the Saccharomyces cerevisiae MLH1 gene reveals distinct roles for Mlh1p in meiotic crossing over and in vegetative and meiotic mismatch repair

Abstract

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.

FIG. 1.

Functional organization of Mlh1p (A) and Pms1p (B). The vertical lines indicate the amino acid positions of the MLH1 alanine scan mutations, with the height of each line corresponding to the mutation frequency relative to the wild-type residue as measured in a canavanine resistance patch assay (Materials and Methods). The numbers directly above the vertical lines identify the MLH1 mutations specifically discussed in the text. The arrows indicate the mutation frequency for EAY841 containing pEAA109 (wild-type, 1×) and pRS415 (mlh1Δ, 16×). The amino acid substitutions indicated below Mlh1p and above Pms1p indicate the polymorphisms that exist between the S288C (first letter) and SK1 (second letter) strain backgrounds. The 447CEGT polymorphism corresponds to a four-residue insertion between residues 447 and 448 in the S288C sequence of Pms1p. The white bars indicate conserved ATP binding domain motifs (8), and the hatched areas indicate the Mlh1p-Pms1p interaction domain (45). The asterisks in MLH1 and PMS1 correspond to a residue in MutL which has been implicated in DNA binding (9).

FIG. 2.

Relative mutation rates of mlh1 alleles. Forward mutation to canavanine resistance (A) and hom3-10 reversion (B and C) are presented for the SK1-derived strain (EAY841), and forward mutation to canavanine resistance (D, EAY874) and repeat tract instability (E, EAY774) are presented for the S288C strains. Mutation rates were determined by the method of the median (at least seven cultures per experiment) (38). Presented are the averages and standard deviations of three independent repetitions relative to the wild-type control (mlh1Δ transformed with pEAA109). The rates for the wild-type controls were as follows: 3.2 × 10⁻⁶ (A), 7.2 × 10⁻⁷ (B), 2.3 × 10⁻⁶ (D), and 9.8 × 10⁻⁶ (E). In panel C, all strains contained both the MLH1 experimental plasmid and an ARS-CEN-URA3 plasmid bearing the PMS1 gene from S288C (pJH480). In this experiment, the rate of hom3-10 reversion is presented relative to that of EAY841 containing pEAA109 and pJH480 (2.5 × 10⁻⁷).

FIG. 3.

Two-hybrid and biochemical analysis of MLH1 mutations. (A) Two-hybrid interactions between lexA-mlh1 and GAL4-PMS1, -MLH3, -EXO1, and -SGS1 fusion constructs. Plates overlaid with paper filters were incubated at 30°C, and expression of the lacZ reporter gene was determined (Materials and Methods). (B) Purification of Msh2p-Msh6p and mutant Mlh1p-Pms1p complexes. Approximately 4 pmol of Msh2p-Msh6p, Mlh1p, and the indicated Mlh1p-Pms1p complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% gel). Lane M, size standards (in kilodaltons). The gels were visualized with Coomassie blue. (C) Ternary complex formation involving Msh2p-Msh6p, Mlh1p-Pms1p, and +1 mismatch substrate. Binding reactions and gel retardation assays were performed as described in Materials and Methods.

FIG. 4.

Distribution of viable spores in SK1 tetrads. The distribution of classes containing 0 to 4 spores per tetrad is presented. Above each graph is the overall spore viability (spo. viab.) and the total number of tetrads dissected from each genotype.