ROLE OF MISMATCH REPAIR IN MAINTAINING GENOME STABILITY

本项目旨在了解真核错配修复（MMR）蛋白在纠正DNA复制错误和处理重组事件中的作用。在从大肠杆菌到人类的生物体中，MMR在纠正DNA复制过程中发生的错误整合错误和滑移事件中起着重要作用。在大肠杆菌中，MutS和MutL在MMR中起关键作用；MutS结合错偶，MutL与mts -错偶复合物相互作用。在酵母和高等真核生物中已经发现了MutS （Msh）和MutL （Mlh）的同源物。对细菌、酵母和人类的研究也表明，MMR通过防止同源DNA序列之间的重组，在维持染色体稳定性方面发挥着重要作用。在真核生物中，在DNA复制和基因重组过程中，MMR蛋白识别错配底物并将信息传递给下游修复因子的分子机制尚不清楚。这些问题将通过以下方法在酿酒酵母中得到解决：1。1994年提交的目标是确定条件msh2突变，因为它们可以揭示途径中的中间步骤，并为第二位点抑制因子筛选提供关键试剂。研究人员将对6个msh2和4个mlh1条件MMR突变进行基因抑制分析，以确定msh2和mlh1以及新的下游MMR因子之间的相互作用。2. 生化方法将用于检查Msh2p-Msh6p （mutS同源）错配识别复合体与其他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msh2和msh6突变。3. mlh1突变体在MMR和杂交中表现出明显的缺陷。为了验证MLH1在这些过程中发挥独特和不同作用的观点，我们将对MLH1进行突变，目的是确定影响一个而不是两个过程的MLH1突变。4. 为了了解MMR因子如何被招募到重组中间体中，我们正在使用ChIP（染色质交联，免疫沉淀和PCR）研究Msh2p与DNA的关联。该程序将在野生型和重组缺陷突变体中使用质粒和染色体重组试验进行。

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.