DNA Expansion and Mismatch Repair

DNA 扩增和错配修复

• 批准号: 9978826
• 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
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978826
• 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; Estrogen receptor positive; 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

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 该提议的总体目的是剖析cag发夹结合的悖论机制 将原本正常的MMR复合物转换为突变机。哺乳动物细胞已经发展 软化的DNA修复系统以纠正遗漏或损坏的碱基和外去循环。出奇， 真核生物不匹配的识别综合体MSH2/MSH3无法充当基因组的监护人 引起CAG扩张，亨廷顿氏病（HD）的致命突变和其他20多个 神经退行性疾病。在此提案中，我们专注于导致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水解未能拆卸信号，并且（2）识别 由MSH2-MSH3发病蛋白招募的核酸酶，该核酸酶将回路纳入复式DNA 完成扩展。在AIM 1A中，我们将生成两个用于MSH2-的“功能分离”突变体Ki小鼠 MSH3，在每个亚基中结合ATP，但在一个或另一个亚基中缺乏ATP水解功能。如果水解损失 特定亚基中的活性减弱了扩张，然后突变需要ATPase活性 亚基。在AIM 1B中，我们将解决与维修胜任的MSH2-MSH3的晶体结构（CA）4环或 到耐修复的CAG发夹。确定的是核苷酸结合的MSH2-的结构扰动 MSH3复合物可防止正确拆除发夹循环。在AIM 2中，我们将确定规范和非 - 规范性核酸内切酶机械促进了发夹循环并完成扩展。 为了识别非典型机械，我们将开发技术以特定于网站的内切核酸酶 在扩展过程中切开CAG道的循环的“捕获”。用CRIS插入DNA网站 提供了一个工程的着陆垫来瞄准工程的APEX2融合蛋白。后一个修饰符 位置紧密的蛋白质与生物素合作伙伴，可以在链霉亲和素板上捕获。我们将测试这些 当MSH2-MSH3与CAG发夹绑定时，指示“在trans”上被错过了。 总体而言，提出的实验为小分子开发铺平了道路，以恢复循环删除 改变发夹DNA结构或蛋白质构象。