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

PCNA依赖性5->3错配切除机制

• 批准号: 7631308
• 负责人: Guo-Min Li
• 金额: $ 22.72万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7631308
• 关键词: Agreement; Base Pairing; Cells; DNA; Defect; Dependency; Development; Discrimination; Disease; EXO1 gene; Escherichia coli; Excision; Exonuclease; Genes; Genome; Genomic Instability; Goals; HMGB1 gene; Health; Hereditary Nonpolyposis Colorectal Neoplasms; Human; In Vitro; Investigation; Laboratories; Lead; Maintenance; Malignant Neoplasms; Mismatch Repair; Modeling; Molecular; Mus; Pathway interactions; Polymerase; Positioning Attribute; Predisposition; Proteins; Reaction; Reporting; Role; Signal Transduction; Syndrome; System; Work; base; expectation; novel; novel diagnostics; nuclease; reconstitution; spleen exonuclease

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.

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基因的缺陷导致严重的基因组不稳定，并最终导致癌症易感性，包括遗传性非息肉性结直肠癌（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是镍导向的，涉及至少9种蛋白质，包括MutS α、MutS β、MutL α、EXO1、聚合酶δ、PCNA、RFC、RPA和HMGB1。尽管对错配引起的切除的启动进行了广泛的研究，这似乎需要MutS α， PCNA， RPA和EXO1。然而，MutS蛋白的错配识别如何导致距离错配位点数百个碱基对的单链断裂处的链特异性切除尚不清楚。目前关于错配诱发切除的三种模型在需要多少MutS分子和如何识别链识别信号方面存在很大争议。最近，错配引起的切除已被重建。有趣的是，EXO1，一个5'？>3'外切酶，据报道能够双向去除错配；并且exo1催化的5'切除不依赖于PCNA。鉴于PCNA在错配引起的切除中的重要性以及大肠杆菌甲基直接MMR中四种外切酶的参与，我们假设多种核酸酶参与了人类的反应。事实上，我们最近发现了一种新颖的5'？>3‘错配切除途径，至少依赖于MutS α、PCNA、一种新型5’核酸酶和一种刺激因子。本申请提出纯化和表征新型5‘核酸酶和切除刺激因子，并最终在体外使用纯化蛋白重建依赖于pna的5’切除反应。纯化后的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（如EXO1基因）的缺陷与癌症的发展有关，因此确定新型5'切除途径所需的组分将为HNPCC和其他MMR缺陷型癌症综合征提供新的诊断标记。