ROLE OF MISMATCH REPAIR IN MAINTAINING GENOME STABILITY

本计画旨在了解真核生物错配修复蛋白在纠正DNA复制错误和处理重组事件中的作用。 在从E. MMR在纠正DNA复制过程中发生的错误插入错误和滑动事件中起着重要作用。 在大肠在MMR中，MutS和MutL起关键作用; MutS与错配结合，MutL与MutS错配复合物相互作用。 MutS（Msh）和MutL（Mlh）的同源物已经在酵母和高等真核生物中鉴定。 对细菌、酵母和人类的研究也表明，MMR通过阻止同源DNA序列之间的重组，在维持染色体稳定性方面发挥重要作用。MMR蛋白识别错配底物并在DNA复制和遗传重组过程中将此信息传递给下游修复因子的分子机制在真核生物中还不清楚。 这些问题将在酵母S.酿酒酵母通过以下途径：1. 1994年提交的一个目标是鉴定条件msh 2突变，因为它们可以揭示途径中的中间步骤，并为第二位点抑制筛选提供关键试剂。将对6个充分表征的msh 2和4个mlh 1条件性MMR突变进行遗传抑制分析，以确定MSH 2和MLH 1与新的下游MMR因子之间的相互作用。 2.生物化学方法将用于检查Msh 2 p-Msh 6p（mutS同源物）错配识别复合物与其他DNA修复和复制因子之间的相互作用。该分析将利用我们分离的大量显性阴性、条件性和位点特异性msh 2和msh 6突变。 3. mlh 1突变体在MMR和交换中显示出明显的缺陷。 为了测试MLH 1在这些过程中发挥独特作用的想法，我们将诱变MLH 1，目的是鉴定影响一个而不是两个过程的MLH 1突变。 4.为了了解MMR因子是如何被募集到重组中间体中的，我们正在使用ChIP（染色质交联，免疫沉淀和PCR）研究Msh 2 p与DNA的相关性。 将使用质粒和染色体重组试验在野生型和重组缺陷突变体中进行该程序。

This project aims to understand the actions of eukaryotic mismatch repair (MMR) proteins in correcting DNA replication errors and in processing recombination events. In organisms ranging from E. coli to humans, MMR plays an important role in correcting misincorporation errors and slippage events that occur during DNA replication. In E. coli, MutS and MutL play critical roles in MMR; MutS binds to mispairs and MutL interacts with MutS-mispair complexes. Homologs of MutS (Msh) and MutL (Mlh) have been identified in yeast and higher eukaryotes. Studies in bacteria, yeast and humans have also shown that MMR plays an important role in maintaining chromosome stability by preventing recombination between homologous DNA sequences. The molecular mechanisms by which MMR proteins recognize mismatch substrates and transduce this information to downstream repair factors during DNA replication and genetic recombination are not well understood in eukaryotes. These issues will be addressed in the yeast S. cerevisiae through the following approaches: 1. A goal of the 1994 submission was to identify conditional msh2 mutations as they can reveal intermediate steps in a pathway and provide critical reagents for a second site suppressor screen. Genetic suppression analysis will be performed with six well characterized msh2 and four mlh1 conditional MMR mutations to identify interactions between MSH2 and MLH1 and new downstream MMR factors. 2. Biochemical approaches will be used to examine interactions between the Msh2p-Msh6p (mutS homolog) mismatch recognition complex and other DNA repair and replication factors. This analysis will take advantage of a large collection of dominant negative, conditional, and site specific msh2 and msh6 mutations that we isolated. 3. mlh1 mutants show distinct defects in MMR and in crossing over. To test the idea that MLH1 plays unique and distinct roles in each of these processes, we will mutagenize MLH1 with the goal of identifying mlh1 mutations that affect one but not both processes. 4. To understand how MMR factors are recruited to recombination intermediates, we are investigating the association of Msh2p with DNA using ChIP (chromatin crosslinking, immunoprecipitation and PCR). This procedure will be performed in wild type and recombination defective mutants using both plasmid and chromosomal recombination assays.