Inactivation of DNA mismatch repair by increased expression of yeast MLH1.

Mismatch repair (MMR) is an important and powerful mutation avoidance mechanism in prokaryotes and eukaryotes. A major function of MMR proteins is to correct mismatches arising during DNA replication and recombination (reviewed in references 2, 20, 29, and 44). In Escherichia coli, the early steps in MMR require the products of the mutS and mutL genes. These genes are conserved in eukaryotes, which contain multiple MutS and MutL homologs that are proposed to function early in MMR. Repair of mismatches in DNA involves either of two complexes of MutS homologs. The MSH2-MSH6 heterodimer participates in the repair of base-base mismatches and insertion-deletion mismatches involving a small number of nucleotides, while MSH2-MSH3 is responsible mainly for repair of insertion-deletion mismatches involving a larger number of nucleotides. MMR also requires complexes of MutL homologs, three of which have been described for both yeast and humans. One is a heterodimer of Mlh1p and Pms1p in yeast (36) or of MLH1 and PMS2 in humans (26) that has been designated hMutLα. The Saccharomyces cerevisiae Mlh1p-Pms1p heterodimer forms a complex with Msh2p-Msh6p or Msh2p-Msh3p bound to mismatched DNA in vitro (12, 13). This suggests that the Mlh1p-Pms1p heterodimer participates in correcting mismatches recognized by both Msh2p-Msh6p and Msh2p-Msh3p. Yeast Mlh1p has been also shown to interact with two other MutL homologs, Mlh3p and Mlh2p, presumably forming heterodimers (46), and human MLH1 protein has been shown to interact with the human MLH3, a homolog of yeast Mlh3p (27). The yeast Mlh1p-Mlh3p complex was suggested by genetic data to participate in Msh2p-Msh3p-dependent repair of insertion-deletion mispairs (10). A minor role in correcting frameshift intermediates has been also proposed for the yeast Mlh1p-Mlh2p heterodimer (14). Human MLH1 protein also interacts with human PMS1 to form a heterodimer designated hMutLβ (24, 37), although the function of this complex in MMR or other processes remains to be established. Mutations in MMR genes can inactivate MMR and thus strongly elevate spontaneous mutation rates, especially addition and deletion mutations in short, repetitive (microsatellite) sequences. These MMR gene mutations also predispose humans and mice to tissue-specific cancers. In addition to inactivation via gene mutation, MMR activity can also be modulated by changes in expression of MMR genes. For example, hypermethylation of the human MLH1 promoter results in lack of the MLH1 gene expression, loss of MMR activity, and microsatellite instability (16, 18). An increasing number of reports describe a correlation between human MLH1 promoter hypermethylation and sporadic colon, gastric, and endometrial cancers with microsatellite instability (see, for example, references 9, 16, 23, and 45). Conversely, MMR activity can be reduced by increased expression of MMR genes. Thus, methotrexate-resistant human cell lines that overproduce MSH3 have a base substitution mutator phenotype (8). Extracts of these cells are defective in repairing base-base mismatches, and repair can be restored by addition of purified hMSH2-hMSH6 complex. The MMR defect was suggested to result from diminished MSH2-MSH6 levels due to sequestration of MSH2 by excess MSH3 (8, 28). Elevated mutation rates have also been reported in yeast strains overproducing either of two eukaryotic MutL homologs. Expression of the PMS1 gene from a multicopy plasmid in a wild-type yeast strain produced a 10-fold increase in the reversion rate of the hom3-10 frameshift mutation (21). More recently, we found that increased expression of the yeast MLH1 gene produced a strong mutator phenotype for several genetic markers, with the mutation rate approaching that of a mlh1 deletion strain (39). These mutator effects are in contrast to results with the yeast MSH2 gene, which does not confer hypermutability when overexpressed in wild-type yeast strains (7). In this study, we explore the mechanism of the Mlh1p-induced mutator effect. Using hybridization to DNA microarrays, we show that overexpression of MLH1 does not induce changes in expression of other genes involved in DNA repair or replication. While Mlh1p is required to repair replication errors, it also participates in other DNA transactions including recombination and transcription-coupled nucleotide excision repair (17, 22, 46) which can modulate the mutation rate. To determine if the Mlh1p-induced mutator phenotype was due to failure to correct DNA replication errors, here we have examined effects of MLH1 overexpression in strains defective in genes controlling either DNA replication or MMR. The data suggest that the mutator phenotype of MLH1-overexpressing strains results from inactivation of MMR dependent on MSH2, MLH1, and PMS1 genes. We also show that the Mlh1p-induced mutator phenotype can be suppressed by overexpression of PMS1 or by a missense mutation in the MLH1 gene that disrupts Mlh1p interaction with Pms1p. Finally, we show that purified Mlh1p is capable of forming a homodimer in solution, a finding which suggests that MMR in cells overexpressing MLH1 could be disrupted by nonfunctional MMR complexes containing Mlh1p homodimers.