Mechanism of PCNA-dependent 5'->3' Mismatch Excision

项目描述（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基因的缺陷导致严重的基因组不稳定性，并最终导致癌症易感性，包括遗传性非息肉病性结直肠癌（HNPCC）和某些类型的散发性癌症，这一事实强调了MMR的重要性。人类细胞中的MMR是切口导向的，并且涉及至少九种蛋白质，它们是MutS α、MutS β、MutL α、EXO 1、聚合酶δ、PCNA、RFC、RPA和HMGB 1。尽管广泛的调查，启动不匹配引起的切除，这似乎需要MutS α，PCNA，RPA，和EXO 1。然而，MutS蛋白的错配识别如何导致距离错配数百个碱基对的单链断裂处的链特异性切除尚不清楚。目前关于错配引起的切除的三种模型在需要多少MutS分子以及如何识别链辨别信号方面是相当有争议的。最近，由不匹配引起的割礼得到了恢复。有趣的是，EXO 1，一个5 '？>据报道，3'外切核酸酶能够双向去除错配;并且EXO 1催化的5'切除不依赖于PCNA。考虑到PCNA在错配引起的切除中的重要性和大肠杆菌中四种核酸外切酶的参与。大肠杆菌甲基直接MMR，我们假设，多个核酸酶参与了人类的反应。事实上，我们最近发现了一种新的5 '？3'错配切除途径，其至少依赖于MutS α、PCNA、新型5'核酸酶和刺激因子。本申请提出纯化和表征新的5'核酸酶和切除刺激因子，并最终使用纯化的蛋白质在体外重建PCNA依赖性5'切除反应。纯化的系统，然后将用于评估三个有争议的模型，以阐明错配引起切除的分子机制。由于错配切除基因的缺陷，例如，EXO 1基因与癌症发展相关，鉴定新的5'切除途径所需的组分将为HNPCC和其他MMR缺陷癌症综合征提供新的诊断标志物。

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 severe genomic instability and eventually to cancer predisposition, including hereditary non-polyposis colorectal cancer (HNPCC) and certain types of sporadic cancers. MMR in human cells is nick-directed and involves at least nine proteins, which are MutS alpha, MutS beta, MutL alpha, EXO1, polymerase delta, PCNA, RFC, RPA, and HMGB1. Despite extensive investigations on the initiation of mismatch-provoked excision, which appears to require MutS alpha, PCNA, RPA, and EXO1. However, how mismatch recognition by MutS proteins leads to a strand-specific excision at a single-stranded break several hundred base pairs away from the mismatch is not understood. Three current models on mismatch- provoked excision are quite controversial in terms of how many MutS molecules are required and how the strand discrimination signal is recognized. Recently, mismatch-provoked excision has been reconstituted. Interestingly, EXO1, a 5'?>3' exonuclease, was reported to be capable of bi-directional removal of mismatch; and EXO1-catalyzed 5' excision is independent of PCNA. Given the importance of PCNA in mismatch- provoked excision and the involvement of four exonucleases in the E. coli methyl-direct MMR, we hypothesize that multiple nucleases are involved in the human reaction. Indeed, we have recently identified a novel 5'?>3' mismatch excision pathway, which depends on at least MutS alpha, PCNA, a novel 5' nuclease, and a stimulating factor. This application proposes to purify and characterize both the novel 5' nuclease and the excision stimulating factor, and eventually reconstitute the PCNA-dependent 5' excision reaction in vitro using purified proteins. The purified system will then be used to evaluate three controversial models to elucidate the molecular mechanism of mismatch-provoked excision. Since defects in mismatch excision genes, e.g., the EXO1 gene, are associated with cancer development, identifying the components required for the novel 5' excision pathway will provide new diagnostic markers for HNPCC and other MMR deficient cancer syndromes.