Dissection and Reconstitution of Human Mismatch Repair

项目描述（由申请人提供）：本项目的长期目标是了解人类错配修复（MMR）的分子机制及其对人类健康和疾病的影响。MMR基因的缺陷导致基因组不稳定并最终导致癌症易感性，这一事实强调了MMR的重要性。尽管最近已经使用纯化蛋白（包括MutS α、MutL α、EXO1和RPA）重建了人MMR反应的切除步骤，但MMR的许多基本问题仍未得到解答。例如，错配引起的切除如何开始和终止，以及MutL α在MMR中起什么作用尚不清楚。通过重构5'镍导向的人MMR反应，我们发现MutL α负调控错配引起的exo1催化的切除，并在错配去除后立即终止。考虑到EXO1催化的错配切除依赖于MutS α以及EXO1、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是通过这些蛋白质之间的相互作用精确调节的。具体来说，在存在错配的情况下，MutS α优先与EXO1相互作用以促进切除；但是通过EXO1去除不匹配允许EXO1优先与MutL α相互作用，然后终止切除。为了验证这一假设，将构建、表达和纯化一系列破坏EXO1与一个Mut蛋白而不破坏另一个Mut蛋白相互作用的EXO1、MutS α、MutL α突变蛋白。这些突变蛋白将在体外重组系统中进行测试，以确定它们在起始和终止处执行错配引起的切除的能力。对每个反应中切除中间体的分析将揭示MutS α、MutL α和EXO1之间的相互作用如何调节切除反应的特定步骤。最后，为了了解MMR系统如何有效地终止错配引起的切除，将精确确定具有不同序列内容和距离将错配和链断裂分开的异双工的切除终止位点。除了揭示错配引起的切除的分子机制外，本研究将为理解mlh1空突变体中关于5'定向MMR、减数分裂细胞凋亡和抗重组等许多令人费解的现象提供重要线索。

DESCRIPTION (provided by applicant): 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 EXO1 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 different sequence 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 mlh1 null mutants regarding 5' directed MMR, meiotic cell apoptosis, and anti-recombination.