Dissection and Reconstitution of Human Mismatch Repair

本项目的长期目标是了解人类错配修复的分子机制 (MMR)及其对人类健康和疾病的影响。孕产妇死亡率的重要性突出体现在以下事实中： MMR基因中的缺陷导致基因组不稳定并最终导致癌症倾向。尽管 人MMR反应的切除步骤最近已经使用纯化的蛋白质重建， 包括MutS alpha、MutL alpha、EXO 1和RPA，MMR中的许多基本问题仍然存在 无人回应例如，错配引起的切除是如何启动和终止的，MutL在其中扮演什么角色？ MMR中的α作用是未知的。通过5 ′切口导向的人MMR反应的重建， 我们发现MutL α负调节错配引起的EXO 1催化的切除，并终止 它在失配消除后立即发生。考虑到EXO 1催化的错配切除对 MutS α和EXO 1、MutS α和MutL α之间的物理相互作用，我们假设 错配引起的切除通过这些蛋白质之间的相互作用精确地调节。具体到 存在错配时，MutS α优先与EX 01相互作用以促进切除;但 通过EXO 1去除错配允许EXO 1优先与MutL α相互作用， 终止切除。为了验证这一假设，一系列EXO 1，MutS α，MutL α突变蛋白， 将构建、表达破坏EXO 1与一种Mut蛋白相互作用但不破坏另一种Mut蛋白相互作用的重组质粒， 提纯这些突变蛋白将在体外重建系统中测试其执行 在开始和终止时的不匹配引起的切除。中的切除中间体的分析 每个反应都将揭示MutS α、MutL α和EXO 1之间的相互作用如何调节细胞的增殖。 切除反应的特定步骤。最后，为了了解MMR系统终止的效率， 错配引起的切除，具有不同序列的异源双链体的切除终止位点 将精确测定错配和链断裂的含量和间隔距离。在 除了揭示错配引发切除的分子机制外，这项研究还将提供 理解mlh 1无效突变体中关于5 ′定向MMR的许多令人困惑的现象的重要线索， 减数分裂细胞凋亡和抗重组。

The long-term goal of this project is to understand the molecular mechanism of human mismatch repair (MMR) and its impact on human health and disease. The importance of MMR is underscored by the fact that defects in MMR genes lead to genomic instability and eventually to cancer predisposition. Despite the fact that the excision-step of the human MMR reaction has been recently reconstituted using purified proteins, including MutS alpha, MutL alpha, EXO1, and RPA, many fundamental questions in MMR still remain unanswered. For example, how mismatch-provoked excision initiates and terminates, and what role MutL alpha plays in MMR are unknown. Through the reconstitution of the 5' nick-directed human MMR reaction, we found that MutL alpha negatively regulates mismatch-provoked, EXO1-catalyzed excision and terminates it immediately upon mismatch removal. Given the dependency of the EXO1-catalyzed mismatch excision on MutS alpha and physical interactions among EXO1, MutS alpha, and MutL alpha, we hypothesize that mismatch-provoked excision is precisely regulated through interactions among these proteins. Specifically, in the presence of a mismatch, MutS alpha preferentially interacts with EX01 to promote excision; but the removal of the mismatch by EXO1 allows EXO1 to preferentially interact with MutL alpha, which then terminates the excision. To test this hypothesis, a series of EXO1, MutS alpha, MutL alpha mutant proteins that disrupt the interaction of EXO1 with one Mut protein but not the other will be constructed, expressed, and purified. These mutant proteins will be tested in an in vitro reconstitution system for their ability to perform mismatch-provoked excision at the initiation and at the termination. Analysis of the excision intermediates in each reaction will reveal how the interactions among MutS alpha, and MutL alpha, and EXO1 regulate a particular step of the excision reaction. Finally, to understand how efficiently the MMR system terminates mismatch-provoked excision, the excision termination sites for heteroduplexes with differentsequence contents and distances separating the mismatch and the strand break will be precisely determined. In addition to revealing the molecular mechanism of mismatch-provoked excision, this study will provide important clues to understand many puzzling phenomena in mlhl null mutants regarding 5' directed MMR, meiotic cell apoptosis, and anti-recombination.