Mismatch Repair and DNA expansion

错配修复和 DNA 扩增

• 批准号: 7996892
• 负责人: Cynthia Therese McMurray
• 金额: $ 14.16万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-07 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7996892
• 关键词: DNA; Mismatch Repair

DESCRIPTION (provided by applicant): Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extrahelical loops. We have surprisingly found that the eukaryotic mismatch recognition complex, MSH2/MSH3, not only fails to act as a guardian of the genome, but also causes CAG expansion, the lethal mutation underlying Huntington's disease (HD). It is the overall aim of this renewal to build on these discoveries, and to dissect the paradoxical mechanism by which binding of a CAG hairpin converts functional MSH2/MSH3 into a defective machine that causes mutation and disease. Although the molecular details of MutSa function are still incompletely understood, it is clear that some form of conformational coupling between DNA recognition and the nucleotide binding sites plays a central role. Our preliminary data indicate that MSH2/MSH3 binds with high affinity to the CAG hairpin, but binding there inhibits its ATPase activity, alters nucleotide affinity, and prevents translocation along DNA relative to repair competent loops. In order to define how the disease-causing CAG-hairpin diverts the MSH2MSH3 protein from normal repair, it becomes imperative to define the DNA and nucleotide dependent properties of the repair-competent MSH2MSH3-DNA complexes. The biochemical characterization of MSH2/MSH3 has lagged far behind that of MSH2/MSH6, and many parameters are unknown. MSH2/MSH3 is different in the way it recognizes DNA, and has different lesion specificity, and the sites of nucleotide binding in MSH2 and MSH3 subunits have not yet been mapped. Despite key differences, the biochemical properties of MSH2/MSH3 have been primarily extrapolated from MutS and its mammalian homologue, MSH2/MSH6. We propose two specific aims that elucidate the mechanism of uncoupling and how dissociation of DNA binding and ATP hydrolysis in MSH2/MSH3 leads to CAG expansion and disease. In Aim 1, we create a dynamic system for visualizing DNA and nucleotide dynamics in MSH2/MSh3-CAG hairpin complexes. Using combined smFRET and a panel of biochemical methods, We establish a real time system to monitor the relationships between DNA bindings, nucleotide binding and translocation of MSH2/MSh3 on repair competent and repair deficient substrates. In Aim2, we use SAX analysis and limited proteolysis to probe the DNA-induced conformational changes imposed on MSH2/MSH3 by hairpins binding. We will test whether those conformational alterations determine template specificity and influence in protein interactions that divert MSH2/MSh3 function in MMR. These two aims integrate protein biochemistry, nucleotide dynamics, and conformational analysis with in vivo biology in primary animals cells reflecting the disease. These studies will not only provide insights into how mutations in the mismatch repair genes cause expansion and disease, but will also broaden our conceptual framework for DNA damage recognition.

描述（由申请人提供）：哺乳动物细胞已经进化出复杂的DNA修复系统，以纠正错配或受损的碱基和螺旋外环。我们惊奇地发现，真核错配识别复合物MSH 2/MSH 3不仅不能作为基因组的监护者，而且还导致CAG扩增，这是导致亨廷顿病（HD）的致命突变。本更新的总体目标是建立在这些发现的基础上，并剖析CAG发夹的结合将功能性MSH 2/MSH 3转化为导致突变和疾病的缺陷机器的矛盾机制。虽然MutSa功能的分子细节仍不完全清楚，但很明显，DNA识别和核苷酸结合位点之间的某种形式的构象偶联起着核心作用。我们的初步数据表明，MSH 2/MSH 3以高亲和力与CAG发夹结合，但在那里结合抑制其ATP酶活性，改变核苷酸亲和力，并防止相对于修复能力环的沿着DNA易位。为了确定致病CAG-发夹如何使MSH 2 MSH 3蛋白从正常修复转移，必须确定有修复能力的MSH 2 MSH 3-DNA复合物的DNA和核苷酸依赖性。MSH 2/MSH 3的生化特性远远落后于MSH 2/MSH 6，许多参数未知。MSH 2/MSH 3识别DNA的方式不同，具有不同的损伤特异性，并且MSH 2和MSH 3亚基中的核苷酸结合位点尚未被定位。尽管存在关键差异，但MSH 2/MSH 3的生物化学性质主要是从MutS及其哺乳动物同源物MSH 2/MSH 6推断的。我们提出了两个具体的目标，阐明解偶联的机制，以及如何在MSH 2/MSH 3中的DNA结合和ATP水解的解离导致CAG扩增和疾病。在目的1中，我们创建了一个动态系统，用于可视化MSH 2/MSH 3-CAG发夹复合物中的DNA和核苷酸动力学。我们建立了一个真实的时间监测系统，利用smFRET技术和一系列生物化学方法，监测MSH 2/MSH 3在有修复能力和修复缺陷的底物上的DNA结合、核苷酸结合和易位之间的关系。在Aim 2中，我们使用SAX分析和有限的蛋白水解来探测通过发夹结合对MSH 2/MSH 3施加的DNA诱导的构象变化。我们将测试这些构象改变是否决定模板特异性和影响蛋白质相互作用，转移MSH 2/MSH 3在MMR中的功能。这两个目标将蛋白质生物化学、核苷酸动力学和构象分析与反映疾病的原代动物细胞中的体内生物学相结合。这些研究不仅将深入了解错配修复基因中的突变如何导致扩张和疾病，还将拓宽我们对DNA损伤识别的概念框架。