Inactivation of DNA mismatch repair by increased expression of yeast MLH1

Inactivation of DNA mismatch repair by mutation or by transcriptional silen cing of the MLH1 gene results in genome instability and cancer predispositi on. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mel. Cell. Biol . 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also r esult from increased expression of yeast MLH1. Here we investigate the mech anism of this mutator effect. Hybridization of poly(A)(+) mRNA to DNA micro arrays containing 96.4% of yeast open reading frames revealed that MLH1 ove rexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1 overexpression strongly enhanced spontaneo us mutagenesis in yeast strains with defects in the 3'-->5' exonuclease act ivity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1. This su ggests that overexpression of MLH1 inactivates mismatch repair of replicati on errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense muta tion in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ul tracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K-d of 3.14 muM, 36-fold higher than that for Ml h1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.