DNA Expansion and Mismatch Repair

DNA 扩增和错配修复

• 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
• 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

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.

摘要 这一建议的总体目标是剖析CAG发夹结合的矛盾机制， 将正常的MMR复合体转化为突变机器。哺乳动物细胞进化出 复杂的DNA修复系统，以纠正错配或损坏的碱基和螺旋外环。令人惊奇的是， 真核错配识别复合物MSH 2/MSH 3不能作为基因组的监护人， 导致CAG扩增，这是导致亨廷顿病（HD）和其他20多种疾病的致命突变。 神经退行性疾病在这个建议中，我们重点关注两个关键的诱变步骤，导致 突变：我们将（1）确定为什么MSH 2-MSH 3中的ATP水解不能去除信号环，以及（2）鉴定 由MSH 2-MSH 3-发夹复合物募集的核酸内切酶，其将环并入双链DNA中 完成扩张。在目标1A中，我们将产生两个“功能分离”突变KI小鼠用于MSH 2- MSH 3，其在每个亚基中结合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 如果一个特定亚基的活性减弱了扩增，那么突变就需要ATP酶活性， 亚单位在目标1B中，我们将解决MSH 2-MSH 3的晶体结构结合到修复能力（CA）4环或 修复困难的CAG发夹确定的是核苷酸结合的MSH 2中的结构扰动， MSH 3复合物，阻止发夹环的正确去除。在目标2中，我们将确定规范和非规范 典型的内切核酸酶机制，其促进发夹环的掺入并完成扩增。 为了识别非经典机制，我们将开发用于核酸内切酶位点特异性捕获的技术 在扩张期间在CAG道处切割环的“动作中被捕获”。用CRISPR插入DNA位点 提供了用于靶向工程化APEX 2融合蛋白的工程化着陆垫。后者修改 与生物素紧密定位的蛋白质配偶体，其可以在链霉亲和素板上捕获。我们将测试这些 当MSH 2-MSH 3与CAG发夹结合时，指令被误解为“反式”切口。 总的来说，所提出的实验为小分子开发铺平了道路，以恢复环的去除。 改变发夹DNA结构或蛋白质构象。