Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations

Abstract

Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer. In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1 function. First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation. Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function. Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis. Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations. Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented. Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain. This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele.

FIG. 1

Missense mutations in MLH1. Alignment of the amino acid sequences of MutL homologs is shown. hMLH1, human MLH1; yMLH1, S. cerevisiae Mlh1p; rMLH1, Rattus norvegicus MLH1; EcMutL, E. coli MutL; StMutL, Salmonella typhimurium MutL; HexB, Streptococcus pneumoniae HexB. Sequences are from SWISS-PROT or GenBank databases. Amino acids that are identical for at least five proteins are in bold. Boxes indicate positions where amino acid changes were found in HNPCC patients and made in yeast MLH1 in this study. The amino acid substitutions are shown below the alignments.

FIG. 2

MLH1 allele status in Lys⁺ revertants of the MLH1/mlh1Δ strain. (A) Locations of primers used for PCR amplification of the wild-type and mlh1Δ::LEU2 alleles. Open box, MLH1 open reading frame; solid box, the LEU2 gene replacing 230 bp of upstream and 300 bp of MLH1 coding region (36). Arrows indicate locations of primers. (B) PCR analysis of the MLH1 locus in Lys⁺ revertants. Lane 1, MLH1/MLH1 diploid; lane 2, MLH1/mlh1Δ diploid; lanes 3 to 9, Lys⁺ revertants obtained in the wild-type MLH1/MLH1 strain; lanes 10 to 16, Lys⁺ revertants with normal mutability obtained in the MLH1/mlh1Δ strain; lanes 17 to 23, Lys⁺ revertants with a mutator phenotype obtained in the MLH1/mlh1Δ strain. Sizes of the amplified fragments (in base pairs) are shown on the right.

FIG. 3

Effect of bleomycin treatment on Lys⁺ reversion in diploid strains. The analysis was performed as described in Materials and Methods.

FIG. 4

Putative locations of MLH1 missense mutations based on the structure of E. coli MutL. In the ribbon diagram of the 40-kDa N-terminal fragment of E. coli MutL protein (1), the four conserved ATP-binding motifs are shown in red, and the amino acids homologous to residues in yeast Mlh1p where the missense mutations were made are marked with black balls. Amino acid residue numbers for E. coli MutL are shown first, with the numbers for homologous, wild-type yeast residues given in parentheses.