DNA Expansion and Mismatch Repair

摘要 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 MMR 复合体转化为突变机器。哺乳动物细胞已经进化 复杂的 DNA 修复系统可纠正错误配对或损坏的碱基和螺旋外环。令人惊讶的是， 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 神经退行性疾病。在本提案中，我们重点关注导致 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，以及 (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 完成扩张。在目标 1A 中，我们将为 MSH2 生成两只“功能分离”突变 KI 小鼠 MSH3，在每个亚基中结合 ATP，但在其中一个亚基中缺乏 ATP 水解功能。如果失去水解作用 特定亚基中的活性减弱了扩增，则突变需要该亚基中的 ATP 酶活性 亚基。在目标 1B 中，我们将解析与修复能力 (CA)4 环结合的 MSH2-MSH3 的晶体结构或 到抗修复的 CAG 发夹。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，我们将识别规范的和非规范的 典型的核酸内切酶机制，促进发夹环的掺入并完成扩张。 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 在扩张过程中“陷入”切开 CAG 道环的过程中。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。后者修改 与生物素紧密结合的蛋白质伙伴，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。

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.