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

1.我们建立了人错配修复基因hMLH 1和hMSH 2在酿酒酵母中的表达系统。我们还构建了一系列具有错配修复缺陷的酵母菌株。当hMLH 1 cDNA在错配修复缺陷型mlhl菌株中表达时，转化子表现出部分抑制突变子表型。当hMLH 1 cDNA也在错配修复-精通菌株中表达时，由于hMLH 1的显性突变子效应，转化子表现为突变子表型。利用后一种表型，我们分析了27个hMLH 1序列，包括21个氨基酸替换，2个无义突变，2个读码框内缺失和2个羧基端移码突变。其中，25个变体对显性增变因子效应没有影响，表明这些hMLH 1是致病性突变。报告为错义突变的两个变体保留了指示显性增变效应的能力，表明这些变体可能是多态性。报告为多态性的其余两种变体保留了显性增变效应，表明这种类型的测定可能检测到未知的功能丧失突变。本研究建立的检测方法有助于更好地了解HNPC的遗传学特征.根据HNPCC国际合作组（ICG-HNPCC）的突变数据库和我们通过这项联合研究对未发表的突变进行的国际调查，已记录了近200种不同的hMSH 2和hMLH 1突变。其中，约10%的hMSH 2突变和30%的hMLH 1突变是错义突变，这表明有相当一部分HNPCC突变需要澄清其致病性的功能意义，以区分非致病性多态性。

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to the muation data base of the International Collaborating Group of HNPCC (ICG-HNPCC) and our international survey of unpublished muatations through this joint study, nearly 200 different hMSH2 and hMLH1 mutations have been documented.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.