MISMATCH REPAIR AND MAINTAINING GENOME STABILITY

本项目的目标是了解错配修复在细胞凋亡中的作用。 维持S.啤酒。在从 E.错配修复已被证明在人类感染大肠杆菌过程中起着重要作用。 在预防自发性点突变以及插入和 重复DNA序列中的缺失突变。在人类中， 错配修复与遗传性非息肉病直接相关 结肠直肠癌，一种被认为影响多达0.5%的人的疾病 人口。在E. coli和革兰氏阳性菌S.酿酒酵母暗示 错配修复在防止染色体 通过排斥含有碱基的重组中间体进行的重排 对不匹配。在大肠大肠杆菌中，体外错配修复系统已被 使用纯化的组分重建。虽然该系统提供了 有很多关于修复机制的信息，但它没有提到DNA 底物在分子水平上被识别或错配修复如何 蛋白质起调节基因重组事件的作用。这些问题将 可通过对以下物质进行详细的遗传和生化分析来解决 S.酿酒酵母MSH2（mutS同源物#2）。MSH2基因高度同源 mutS是E. coli mutHLS错配修复系统 在体外结合碱基对错配。初步研究显示 Msh2是S.啤酒，是 参与预防自发性点突变、插入和缺失 高度重复DNA序列突变，并调节基因 转换区域。 将对MSH2基因进行突变分析，以确定 显性和条件突变体。将对这些突变体进行 对许多事件频率的影响，包括自发性 重复DNA序列的突变、缺失和插入，有丝分裂 以及在多个同源和异位位点的减数分裂重组，和 染色体重排。将对突变体进行生化分析 与含有错配的DNA底物结合并与 其它失配修复组分。 然后将显性和条件突变体用于基因抑制 分析.该分析将在有丝分裂和减数分裂中进行 分析，希望能鉴定出特异性的抑制基因。 使用特异性msh2突变的合成致死性筛选将被 以及一项旨在查明 在重组缺陷、错配修复中致死的突变 这类突变体可能参与标记DNA 链特异性修复。 在两个S.酿酒酵母和E.大肠杆菌的研究表明 错配修复参与防止染色体重排。这 假设将在S.酿酒酵母使用新的和 已建立的测定法。这些分析将用于确定msh2是否 和其它错配修复突变体影响基因转化的频率 在有丝分裂和减数分裂中进行同源和 位于同源或异位位置的同源序列。在 此外，这些突变对一个新的非法 将测试重组分析。

The goal of this project is to understand the role of mismatch repair in maintaining genome stability in S. cerevisiae. In organisms ranging from E. coli to humans, mismatch repair has been shown to play an important role in preventing spontaneous point mutations as well as insertion and deletion mutations in repetitive DNA sequence. In humans, defects in mismatch repair have been direct correlated to hereditary non-polyposis colorectal cancer, a disease that is thought to affects as much as 0.5% of the population. Additional studies in E. coli and S. cerevisiae suggest that mismatch repair plays an important role in preventing chromosomal rearrangements by rejecting recombination intermediates that contain base pair mismatches. In E. coli, an in vitro mismatch repair system has been reconstructed using purified components. While this system has provided much information regarding repair mechanisms, it has not addressed how DNA substrates are recognized at the molecular level or how mismatch repair proteins act to modulate genetic recombination events. These issues will be addressed by undertaking a detailed genetic and biochemical analysis of S. cerevisiae MSH2 (mutS homolog #2). The MSH2 gene is highly homologous to mutS, a critical component of the E. coli mutHLS mismatch repair system that binds to base pair mismatches in vitro. Initial studies have shown that Msh2 is the major mismatch recognition protein in S. cerevisiae, is involved in preventing spontaneous point mutations, insertion and deletion mutations in highly repetitive DNA sequences, and modulates gene conversion tracts. Mutational analysis will be performed on the MSH2 gene to identify dominant and conditional mutants. These mutants will be examined for their effect on the frequency of a number of events, including spontaneous mutations, deletions and insertions at repetitive DNA sequences, mitotic and meiotic recombination at a number of homologous and ectopic sites, and chromosomal rearrangements. Mutants will be analyzed biochemically for binding to DNA substrates containing mismatches and for interaction with other mismatch repair components. Dominant and conditional mutants will then be used in genetic suppressor analysis. This analysis will be performed in both mitotic and meiotic assays, with the hope that specific suppressor will be identified. Synthetic lethality screens using specific msh2 mutations will be initiated, as well as a synthetic lethality screen aimed at identifying mutations that are lethal in recombination deficient, mismatch repair proficient strains, such mutants could be involved in marking DNA for strand specific repair. Studies performed in both S. cerevisiae and E. coli have suggested that mismatch repair in involved in preventing chromosomal rearrangements. This hypothesis will be tested in S. cerevisiae using both novel and established assays. These assays will be used to determine whether msh2 and other mismatch repair mutants affect the frequency of gene conversion and crossing over in both mitosis and meiosis for homologous and homologous sequences placed at either homologous or ectopic positions. In addition the effect of these mutations on a novel illegitimate recombination assay will be tested.