Recognition and Repair of Mismatched DNA by MutY

描述(申请人提供)：E.coliMutY及其哺乳动物同源物(MYH)通过从OG：A错配中去除错误结合的腺嘌呤残基，在防止与7，8-二氢-8-氧-2‘-脱氧鸟苷(OG)相关的突变方面发挥重要作用。最近，编码人类MutY(HMYH)的基因突变与结直肠癌之间的直接关联被发现。这突出了碱基切除修复和OG修复的重要性：A错配在预防癌变中的作用。我的实验室已经制定了一项严谨和多方面的研究计划，旨在提供对MutY和hMYH功能特性的详细了解。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与结直肠癌的关系，以及继续了解这种独特的BER糖基酶错配识别的复杂特征，我们在本次研究拨款申请中提出如下建议：(1)我们将确定与结直肠癌相关的hMYH变异体的功能特性。具体地说，这将涉及分析OG的修复：细菌和哺乳动物细胞中一组具有代表性的hMYH变体的错配。(2)我们将评估这组具有代表性的hMYH变异体的腺嘌呤糖基酶活性，以确定OG：A修复是否由于酶的固有腺嘌呤去除活性的缺陷所致。AIMS 1和AIMS 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中涉及的具体步骤：利用底物和底物类似物的荧光光谱，通过MutY识别错配和腺嘌呤。这将使我们能够揭示OG和A在最初的碱基对识别中所涉及的重要特征，以及OG的识别是如何耦合到腺嘌呤的排泄和去除的。这方面将利用我们之前描述过的底物类似物，并建立在我们为分析MutY而开发的动力学框架上。(4)通过X射线结晶学研究具有过渡态和底物类似物双链体的嗜热脂肪芽孢杆菌MutY的结构特征，为腺嘌呤切除的机理提供深入的研究。

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.