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 修复系统来纠正错误配对或损坏的碱基和螺旋外环。我们惊奇地发现，真核错配识别复合体 MSH2/MSH3 不仅不能充当基因组的守护者，而且还会导致 CAG 扩展，这是亨廷顿病 (HD) 的致命突变。此次更新的总体目标是在这些发现的基础上，剖析 CAG 发夹的结合将功能性 MSH2/MSH3 转化为导致突变和疾病的有缺陷机器的矛盾机制。尽管 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某种形式的构象耦合起着核心作用。我们的初步数据表明，MSH2/MSH3 以高亲和力与 CAG 发夹结合，但在那里的结合会抑制其 ATP 酶活性，改变核苷酸亲和力，并防止相对于修复能力环沿 DNA 易位。为了确定致病的 CAG 发夹如何使 MSH2MSH3 蛋白从正常修复中转移，必须确定具有修复能力的 MSH2MSH3-DNA 复合物的 DNA 和核苷酸依赖性特性。 MSH2/MSH3的生化表征远远落后于MSH2/MSH6，许多参数未知。 MSH2/MSH3识别DNA的方式不同，具有不同的病变特异性，并且MSH2和MSH3亚基中核苷酸结合的位点尚未被定位。尽管存在重大差异，MSH2/MSH3 的生化特性主要是从 MutS 及其哺乳动物同源物 MSH2/MSH6 推断出来的。我们提出了两个具体目标，以阐明解偶联机制以及 MSH2/MSH3 中 DNA 结合解离和 ATP 水解如何导致 CAG 扩张和疾病。在目标 1 中，我们创建了一个动态系统，用于可视化 MSH2/MSh3-CAG 发夹复合物中的 DNA 和核苷酸动态。使用组合的 smFRET 和一组生化方法，我们建立了一个实时系统来监测 DNA 结合、核苷酸结合和 MSH2/MSh3 在修复能力和修复缺陷底物上的易位之间的关系。在 Aim2 中，我们使用 SAX 分析和有限蛋白水解来探测发夹结合对 MSH2/MSH3 造成的 DNA 诱导构象变化。我们将测试这些构象改变是否决定了模板特异性以及对 MMR 中 MSH2/MSh3 功能转移的蛋白质相互作用的影响。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的突变如何导致扩增和疾病，还将拓宽我们 DNA 损伤识别的概念框架。