ROLE OF MISMATCH REPAIR IN MAINTAINING GENOME STABILITY

该项目旨在了解真核细胞错配修复(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各种生物中，MMR在纠正DNA复制过程中发生的误整合错误和滑动事件方面发挥着重要作用。在大肠杆菌中，MutS和MutL在MMR中起关键作用，MutS与错配对结合，MutL与MutS-MisPair复合体相互作用。MutS(MSH)和MutL(MLH)的同源物已经在酵母和高等真核生物中被鉴定出来。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，MMR蛋白在DNA复制和基因重组过程中识别错配底物并将此信息转导到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年提交的报告的目标是鉴定条件性MSH2突变，因为它们可以揭示途径的中间步骤，并为第二个位点抑制子筛选提供关键试剂。将对6个特征良好的MSH2和4个MLH1条件性MMR突变进行基因抑制分析，以确定MSH2和MLH1之间的相互作用以及新的下游MMR因子。2.将使用生化方法研究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。这项分析将利用我们分离的大量显性负突变、条件性突变和位点特异性MSH2和MSH6突变。3.MLH1突变体表现出明显的MMR缺陷和互换缺陷。为了测试MLH1在每个过程中发挥独特而独特的作用的想法，我们将对MLH1进行突变，目的是识别影响一个过程而不是两个过程的MLH1突变。4.为了了解MMR因子是如何被招募到重组中间体的，我们利用芯片技术(染色质交联、免疫沉淀和聚合酶链式反应)研究了Msh2p与DNA的关系。这一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分析进行。

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.