Recognition and Repair of Mismatched DNA by MutY

大肠杆菌MutY及其哺乳动物同源物(MYH)在防止突变中发挥重要作用 与7，8-二氢-8-氧代-2‘-脱氧鸟苷(OG)结合，通过去除错误结合的腺嘌呤残基 来自OG：一场不匹配。最近，编码人类MutY的基因突变之间的直接关联 (HMYH)和结直肠癌已经被发现。这突出了碱基切除修复的重要性， 和OG的修复：预防癌变的错配。我的实验室已经研制出一种 严格和多方面的研究计划，旨在提供对功能的详细了解 MutY和hMYH的性质。重要的是，我们对hMYH变体的功能分析对于 建立与结直肠癌的联系。为了加深对两国关系的理解 HMYH与结直肠癌之间的关系，以及继续了解HMYH与结直肠癌的复杂特征 这种独特的BER糖基酶的错配识别，我们在这项研究拨款中提出了以下建议 应用：(1)我们将确定与以下相关的hMYH变体的功能特性 结直肠癌。具体地说，这将涉及分析OG的修复：一名代表的错配 细菌和哺乳动物细胞中的一组hMYH变体。(2)我们将测定腺嘌呤糖基酶 这组具有代表性的hMYH变异的活性，以确定OG：A修复是否由于 该酶固有的腺嘌呤去除活性存在缺陷。目标1和目标2将提供重要的 关于这些变异与结直肠癌关系的信息。(3)我们将深入了解 OG中涉及的具体步骤：使用MutY进行错配识别和腺嘌呤移除 底物和底物类似物的荧光光谱。这将使我们能够揭示 OG和A在初始碱基对识别中的重要特征，以及OG如何识别 与腺嘌呤挤出和去除相结合。这方面将利用我们已有的底物类似物 并建立在我们为分析MutY而开发的动力学框架上。(4) 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。 具有过渡态和含底物类似物双链的嗜热脂肪芽孢杆菌MutY 射线结晶学。

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.