Joint study on the DNA mismatch repair system : Functional analysis of the DNA mismatch repair genes using Saccharomyces cerevisiae.

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1. We have developed the expression system of human mismatch repair genes, hMLH1 and hMSH2 in Saccharomyces cerevisiae. We have also constructed a series of yeast strains with mismatch repair defect. When hMLH1 cDNA was expressed in mismatch repair-deficient mlhl strain, the transformants demonstrated to suppress partially the mutator phenotype. When hMLH1 cDNA was also expressed in mismatch repair-proficient strain, the transformants demonstrated to be mutator phenotype, due to dominat mutator effect of the hMLH1. Using the later phenotype, we have analyzed 27 hMLH1 sequenceari ants including 21 amno acid substitutions, 2 nonsense mutations, 2 in-frame deletions and 2 carboxy-terminal frameshift mutations. Among these, 25 variants showed no effect on the dominant mutator effect, indicating that these hMLH1 are pathogenic mutations. Two variants reported as missense mutations retained the ability to indicate dominant mutator effect, suggesting that these variants may be polymorphisms. Remaining two variants reported as polymorphisms retained the dominant mutator effect, suggesting that this type of assay has potentially detect unknown loss-of-function mutations. The assay developed in this study could be helpful for better understanding of genetics of HNPCC.2. According to the muation data base of the International Collaborating Group of HNPCC (ICG-HNPCC) and our international survey of unpublished muatations through this joint study, nearly 200 different hMSH2 and hMLH1 mutations have been documented. Among these, approxymately 10% of hMSH2 mutations and 30% of hMLH1 mutations were missense mutations, indicating that there is a significant fraction of HNPCC mutations which need to clarify their functional significance on pathogenicity in order to discriminate from non-pathogenic polymorphisms.