Recognition and Repair of Mismatched DNA by MutY

E.大肠杆菌MutY及其哺乳动物同源物（MYH）在防止突变中起重要作用 通过去除错误掺入的腺嘌呤残基与7，8-二氢-8-氧代-2 '-脱氧鸟苷（OG）相关 OG：A mismatches.最近，编码人类MutY的基因突变之间的直接相关性 （hMYH）和结直肠癌的关系。这突出了碱基切除修复的重要性， 和修复OG：A错配在预防癌变中的作用。我的实验室开发了一种 严格和多方面的研究计划，旨在提供详细的了解功能 MutY和hMYH的性质。重要的是，我们对hMYH变异体的功能分析对于 与结直肠癌的联系。为了进一步了解 hMYH和结直肠癌之间的关系，以及继续我们对 通过这种独特的BER糖基化酶的错配识别，我们在这项研究中提出了以下建议 应用：（1）我们将确定hMYH变异体的功能特性，这些变异体与以下相关： 结肠直肠癌具体地说，这将涉及分析修复OG：A错配的代表 一组细菌和哺乳动物细胞中的hMYH变体。(2)我们将评估腺嘌呤糖苷酶 这一相同的代表性hMYH变体组的活性，以确定减少的OG：A修复是否是由于 酶的固有腺嘌呤去除活性的缺陷。目标1和2将提供重要的 关于这些变异与结直肠癌的关系的信息。（3）我们将提供深入了解 OG中涉及的具体步骤：使用MutY的A错配识别和腺嘌呤去除 用底物和底物类似物进行荧光光谱分析。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 与腺嘌呤的排出和去除有关。这方面将利用底物类似物，我们有 先前表征的，并建立在我们已经开发的用于分析MutY的动力学框架上。（四） 我们将通过研究腺嘌呤切除的结构特性来深入了解腺嘌呤切除的机制。 使用X-10的具有含有过渡态和底物类似物的双链体的嗜热脂肪芽孢杆菌MutY 射线晶体学

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 recogntion 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.