DNA Expansion and Mismatch Repair

DNA 扩增和错配修复

基本信息

批准号：
9766311
负责人：
Cynthia Therese McMurray
金额：
$ 71.69万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9766311
关键词：
ATP Hydrolysis ATP phosphohydrolase Acids Address Attenuated Binding Biological Biotin CAG repeat Cells Chimeric Proteins Clip Clustered Regularly Interspaced Short Palindromic Repeats Complex Crystallization DNA DNA Repair DNA Structure DNA-Directed DNA Polymerase Data Development Disease Engineering Ethers Excision Failure Genetic Genome Germ-Line Mutation Homologous Gene Huntington Disease Hydrolysis Instruction Knock-in Knock-in Mouse MSH2 gene MSH3 gene Mammalian Cell Mediating Mismatch Repair Molecular Conformation Mus Mutation Neurodegenerative Disorders Nucleotides Oranges Outcome Pathway interactions Process Protein Conformation Proteins Repair Complex Resistance Series Side Signal Transduction Site Streptavidin Structure System Technology Testing Trinucleotide Repeats base complex R endonuclease experimental study in vivo mutant nuclease prevent recruit repaired seal small molecule

项目摘要

ABSTRACT It is the overall aim of this proposal to dissect the paradoxical mechanism by which the binding of a CAG hairpin converts an otherwise normal MMR complex into a mutational machine. Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extrahelical loops. Surprisingly, the eukaryotic mismatch recognition complex, MSH2/MSH3, fails to act as a guardian of the genome and causes CAG expansion, the lethal mutation underlying Huntington's disease (HD) and more than 20 other neurodegenerative diseases. In this proposal, we focus on the two key mutagenic steps that cause the mutation: we will (1) determine why ATP hydrolysis in MSH2-MSH3 fails to signal loop removal, and (2) identify the endonuclease recruited by the MSH2-MSH3-hairpin complex that incorporates the loop into duplex DNA completing expansion. In Aim 1A, we will generate two “separation-of-function” mutant KI mice for MSH2- MSH3, which bind ATP in each subunit, but lack ATP hydrolytic function in one or the other. If loss of hydrolytic activity in a particular subunit attenuates expansion, then the mutation requires the ATPase activity in that subunit. In Aim 1B, we will solve the crystal structure of MSH2-MSH3 bound to a repair competent (CA)4 loop or to the repair-resistant CAG hairpin. Identified are the structural perturbations in the nucleotide-bound MSH2- MSH3 complex that prevent proper removal of the hairpin loop. In Aim 2, we will identify the canonical and non- canonical endonuclease machinery that facilitates incorporation of the hairpin loop and completes expansion. To identify non-canonical machinery, we will develop technology for site-specific capture of endonucleases “caught in the act” of incising the loops at the CAG tract during expansion. Inserting a DNA site with CRISPR provides an engineered landing pad for targeting an engineered APEX2 fusion protein. The latter modifies closely located protein partners with biotin, which can be captured on streptavidin plates. We will test how these instructions are misinterpreted for “in trans” nicking when MSH2-MSH3 is bound to the CAG hairpin. Collectively, the proposed experiments pave the way for small molecule development to restore loop removalby altering the hairpin DNA structure or the protein conformation.

抽象的本提案的总体目标是剖析 CAG 发夹结合的矛盾机制将正常的错配修复复合体转变为突变机器。复杂的 DNA 修复系统可以纠正错配或损坏的碱基和螺旋外环。真核错配识别复合物 MSH2/MSH3 无法充当基因组的守护者，导致 CAG 扩张、亨廷顿病 (HD) 和其他 20 多种疾病的致命突变在本提案中，我们重点关注导致神经退行性疾病的两个关键诱变步骤。突变：我们将 (1) 确定为什么 MSH2-MSH3 中的 ATP 水解无法发出环路去除信号，并且 (2) 确定由 MSH2-MSH3-发夹复合物招募的核酸内切酶，将环整合到双链 DNA 中在目标 1A 中，我们将为 MSH2 生成两只“功能分离”突变 KI 小鼠 MSH3，其在每个亚基中结合 ATP，但如果失去水解，则其中一个或另一个亚基缺乏 ATP 水解功能。特定亚基中的活性减弱了扩增，那么突变需要该亚基中的 ATP 酶活性在目标 1B 中，我们将解析与修复能力 (CA)4 环或结合的 MSH2-MSH3 的晶体结构。确定了核苷酸结合的 MSH2- 中的结构扰动。 MSH3 复合物会阻止发夹环的正确去除。在目标 2 中，我们将识别典型的和非典型的。经典的核酸内切酶机制，有助于合并发夹环并完成扩张。为了识别非规范机制，我们将开发核酸内切酶位点特异性捕获技术在扩增过程中“陷入”在 CAG 区域切割环的过程中，使用 CRISPR 插入 DNA 位点。提供了一个用于靶向工程 APEX2 融合蛋白的工程着陆垫，后者修饰。位置紧密的蛋白质与生物素结合，可以在链霉亲和素板上捕获，我们将测试它们如何。当 MSH2-MSH3 与 CAG 发夹结合时，说明会被误解为“反式”切口。总的来说，所提出的实验为小分子开发恢复环去除铺平了道路改变发夹 DNA 结构或蛋白质构象。