DNA Expansion and Mismatch Repair

摘要 这项建议的总体目标是剖析CAG发夹的捆绑的矛盾机制 将原本正常的MMR复合体转化为突变机器。哺乳动物细胞已经进化到 复杂的DNA修复系统，以纠正错配或损坏的碱基和螺旋外环。令人惊讶的是， 真核细胞错配识别复合体MSH2/MSH3未能作为基因组的守护者，并 导致CAG扩张，亨廷顿病(HD)和其他20多种疾病的致命性突变 神经退行性疾病。在本提案中，我们将重点放在导致 突变：我们将(1)确定为什么MSH2-MSH3中的ATP水解未能发出去除环的信号，以及(2)确定 由MSH2-MSH3-发夹复合体招募的内切酶，将环合并到双链DNA中 正在完成扩展。在目标1A中，我们将为MSH2产生两个“功能分离”突变KI小鼠- MSH3在每个亚基上与ATP结合，但在其中一个亚基上缺乏ATP水解性功能。如果水解物损失 特定亚基中的活性减弱了扩张，那么突变需要该亚基中的ATPase活性 亚单位。在目标1B中，我们将解决MSH2-MSH3与修复能力(CA)4环或 到不可修复的CAG发夹。确定了核苷酸结合的MSH2-中的结构扰动- MSH3复合体，防止正确移除发夹环。在目标2中，我们将确定规范和非规范 标准的核酸内切酶机器，促进发夹环的结合并完成扩张。 为了识别非正则机制，我们将开发定点捕获核酸内切酶的技术 在扩张过程中，“陷入”切割CAG区域的环的行为。使用CRISPR插入DNA站点 提供针对工程APEX2融合蛋白的工程着陆台。后者修改了 紧密定位的蛋白质与生物素配对，生物素可以在链霉亲和素平板上捕获。我们将测试这些 当MSH2-MSH3与CAG发夹结合时，指令被误解为“In Transs”刻痕。 总的来说，建议的实验为小分子开发恢复环路移除铺平了道路 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.