Recognition and Repair of Mismatched DNA by MutY

MutY 识别和修复不匹配的 DNA

• 批准号: 7105779
• 负责人: SHEILA Sue DAVID
• 金额: $ 21.17万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105779
• 关键词: DNA binding protein; DNA damage; DNA repair; Escherichia coli; N glycosidase; bacterial genetics; bacterial proteins; conformation; deoxyadenosines; deoxyguanosine; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; gene mutation; iron sulfur protein; nucleic acid sequence; nucleoside analog; protein structure function

DESCRIPTION (provided by applicant): E. coli MutY and its mammalian homologues (MYH) play an important role in the prevention of mutations associated with 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removal of misincorporated adenine residues from OG:A mismatches. Recently, a direct correlation between mutations in the gene encoding human MutY (hMYH) and colorectal cancer has been uncovered. This highlights the importance of base-excision repair, and the repair of OG:A mismatches in the prevention of carcinogenesis. My laboratory has developed a rigorous and multifaceted research program aimed at providing a detailed understanding of the functional properties of MutY and hMYH. Importantly, our functional analysis of hMYH variants has been important for establishing the connection to colorectal cancer. In order to further the understanding of the relationship between hMYH and colorectal cancer, as well as continue our understanding of the complex features of mismatch recognition by this unique BER glycosylase, we propose the following in this research grant application: (1) We will determine the functional properties of variants of hMYH that are correlated with colorectal cancer. Specifically, this will involve analyzing the repair of OG:A mismatches by a representative set of hMYH variants in bacterial and mammalian cells. (2) We will evaluate the adenine glycosylase activity of this same representative set of hMYH variants to determine if reduced OG:A repair is due to defects in the intrinsic adenine removal activity of the enzyme. Aims 1 and 2 will provide important information on the relationship of these variants to colorectal cancer. (3 ) We will provide insight into specific steps that are involved in OG:A mismatch recognition and adenine removal by MutY using fluorescence spectroscopy with substrates and substrate analogues. This will allow us to reveal the important features of OG and A that are involved in initial base-pair recognition, and how recognition of OG is coupled to adenine extrusion and removal. This aspect will utilize substrate analogues that we have characterized previously and build upon the kinetic framework we have developed for analyzing MutY. (4) We will provide insight into the mechanism of adenine excision by examining the structural properties of Bacillus stearotheromophilus MutY with transition state and substrate analogue-containing duplexes using X- ray crystallography.

描述（由申请人提供）：大肠杆菌 MutY 及其哺乳动物同源物 (MYH) 通过从 OG:A 错配中去除错误掺入的腺嘌呤残基，在预防与 7,8-二氢-8-氧代-2'-脱氧鸟苷 (OG) 相关的突变中发挥重要作用。最近，人们发现了编码人类 MutY (hMYH) 的基因突变与结直肠癌之间的直接相关性。这凸显了碱基切除修复和 OG:A 错配修复在预防癌发生中的重要性。我的实验室制定了严格且多方面的研究计划，旨在详细了解 MutY 和 hMYH 的功能特性。重要的是，我们对 hMYH 变异的功能分析对于建立与结直肠癌的联系非常重要。为了进一步了解 hMYH 与结直肠癌之间的关系，并继续了解这种独特的 BER 糖基化酶错配识别的复杂特征，我们在本研究资助申请中提出以下建议：（1）我们将确定与结直肠癌相关的 hMYH 变体的功能特性。具体来说，这将涉及分析细菌和哺乳动物细胞中一组代表性 hMYH 变体对 OG:A 错配的修复。 (2) 我们将评估同一组代表性 hMYH 变体的腺嘌呤糖基酶活性，以确定 OG:A 修复减少是否是由于该酶的内在腺嘌呤去除活性缺陷所致。目标 1 和 2 将提供有关这些变异与结直肠癌关系的重要信息。 (3) 我们将深入了解 OG:A 错配识别和 MutY 使用荧光光谱与底物和底物类似物去除腺嘌呤所涉及的具体步骤。这将使我们能够揭示参与初始碱基对识别的 OG 和 A 的重要特征，以及 OG 的识别如何与腺嘌呤挤出和去除相结合。这方面将利用我们之前表征的底物类似物，并建立在我们为分析 MutY 开发的动力学框架之上。 (4) 我们将通过使用 X 射线晶体学检查具有过渡态和底物类似物的双链体的嗜热脂肪芽孢杆菌 MutY 的结构特性，深入了解腺嘌呤切除的机制。