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

描述(申请人提供)：该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基因的缺陷会导致严重的基因组不稳定，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，这一事实突显了MMR的重要性。人类细胞中的MMR是缺口定向的，至少涉及九种蛋白质，分别是MutSα、MutSβ、MutLα、EXO1、聚合酶Delta、增殖细胞核抗原、RFC、RPA和HMGB1。尽管对错配引起的切除的启动进行了广泛的调查，但这似乎需要MutSα、增殖细胞核抗原、RPA和EXO1。然而，MutS蛋白质的错配识别如何导致在远离错配的数百个碱基对的单链断裂上进行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识别信号方面存在相当大的争议。最近，错配引发的切除重新构成了。有趣的是，EXO1是一种5‘？&gt；3’外切酶，据报道能够双向去除错配；EXO1催化的5‘端切除不依赖于增殖细胞核抗原。鉴于增殖细胞核抗原在错配引起的切除中的重要性，以及大肠杆菌甲基直接MMR中四种外切核酸酶的参与，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‘？&gt；3’错配切除途径，它至少依赖于MutSα、增殖细胞核抗原、一种新的5‘核酸酶和一个刺激因子。这项应用旨在纯化和鉴定新的5‘核酸酶和切除刺激因子，并最终用纯化的蛋白在体外重建依赖于增殖细胞核抗原的5’切除反应。然后，纯化系统将用于评估三个有争议的模型，以阐明错配引起的切除的分子机制。由于错配切除基因的缺陷，例如EXO1基因，与癌症的发生有关，识别新的5‘切除途径所需的成分将为HNPCC和其他MMR缺陷型癌症综合征提供新的诊断标记。

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.