Roles for Mismatch Repair Proteins in Maintaining Genome Stability

描述(申请人提供)：错配修复(MMR)将DNA复制的保真度提高约1000倍，方法是切除因误掺入和DNA滑移而导致的新复制链中的错配。MSH蛋白通过与DNA错配结合，然后与MLH蛋白相互作用来招募下游修复因子来启动MMR。MSH和MLH基因的突变使突变率显著增加。MMR因子还阻止不同DNA序列之间的重组，并处理包含非同源的重组中间体 单股末端。这些蛋白质识别错配和信号的机制 DNA复制和重组过程中的下游因素还不是很清楚。此外，遗传背景在决定MMR突变相对于疾病表型的外显性方面的作用还没有深入探讨。这一建议的重点是了解MMR蛋白如何在DNA复制和修复过程中识别错配和信号下游因素，以及遗传背景在决定MMR突变的外显性中的作用。在目标1中，我们将使用全内荧光显微镜分析单个MSH和MLH与DNA相互作用的行为。这些研究将利用之前在实验室中产生的大量MMR突变，旨在区分MSH和MLH蛋白质如何发出MMR下游步骤的竞争模型。在目标2中，我们将使用SNP扫描仪技术来检测MMR突变体的全基因组突变积累。这项工作将使我们能够确定MMR缺陷菌株的实际突变率，并提供信息，帮助癌症研究人员区分对转化为癌症状态至关重要的突变与转化后发生的突变。AIM 3也专注于基因组的稳定性，并在体外和体内呈现 测试MMR组件和SGS1解旋酶之间相互作用的生化方法，以防止不同DNA序列之间的重组。AIM 4概述了旨在了解MMR中基因组不稳定性是如何由遗传不亲和性引起的实验。这项工作将提供模型来解释遗传背景如何影响人类的疾病外显性。此外，它还提供了新的工具来识别DNA修复途径中的遗传相互作用，总体目标是了解癌症易感性和在DNA修复中发挥作用的分子途径。

DESCRIPTION (provided by applicant): Mismatch repair (MMR) improves the fidelity of DNA replication by about 1000 fold by excising mismatches in the newly replicated strand arising from mis-incorporation and DNA slippage. MSH proteins initiate MMR by binding to DNA mismatches and then interacting with MLH proteins to recruit downstream repair factors. Mutations in MSH and MLH genes confer significant increases in mutation rate. MMR factors also prevent recombination between divergent DNA sequences, and process recombination intermediates containing nonhomologous single-stranded ends. The mechanisms by which these proteins identify mismatches and signal downstream factors during DNA replication and recombination are not well understood. In addition, the role of genetic background in determining the penetrance of MMR mutations with respect to disease phenotype has not been explored in depth. This proposal is focused on understanding how MMR proteins identify mismatches and signal downstream factors during DNA replication and repair, and the role of genetic background in determining the penetrance of MMR mutations. In Aim 1 we will analyze the behavior of single MSH and MLH complexes interacting with DNA using total internal fluorescence microscopy. These studies will take advantage of a large number of MMR mutants generated previously in the lab and are aimed at distinguishing between competing models for how MSH and MLH proteins signal downstream steps in MMR. In Aim 2 we will use SNP scanner technology to examine genome-wide mutation accumulation in MMR mutants. This work will allow us to determine the actual mutation rate in a MMR-defective strain and provide information that should help cancer researchers distinguish mutations critical for transformation to a cancer state from those that occur after transformation. Aim 3 is also focused on genome stability and presents in vitro and in vivo biochemical approaches to test interactions between MMR components and the SGS1 helicase to prevent recombination between divergent DNA sequences. Aim 4 outlines experiments aimed at understanding how genome instabilities arise from genetic incompatibility in MMR. This work will provide models to explain how genetic background contributes to disease penetrance in humans. In addition it offers new tools to identify genetic interactions in DNA repair pathways, with the overall goal of understanding cancer susceptibility and the molecular pathways that function in DNA repair.