DNA DAMAGE REPAIR BY MUTYH AND MUTYH VARIANTS ASSOCIATED WITH COLORECTAL CANCER

 描述(申请人提供)：MUTYH糖基酶因发现遗传的MUTYH变异与结直肠癌(CRC)之间的相关性而备受关注。利用稳定前动力学和含有2‘-脱氧腺苷类似物的不可切割寡核苷酸，我们提供了在结直肠癌中发现的两个MUTYH错义变异的功能缺陷的信息，这两个错义变异是建立MUTYH变异与结直肠癌之间联系的关键(现在被称为MUTYH相关息肉病或MAP)。MUTYH通过从8-氧-7，8-二氢-2‘-脱氧鸟苷(OG：A错配)中去除错误结合的腺嘌呤残基来防止与8-氧-7，8-二氢-2’-脱氧鸟苷(OG)相关的突变，从而导致功能异常的MUTYH导致肿瘤抑制基因(如APC)突变的积累。自从MAP最初被发现以来，已经发现了100多种不同的MUTYH变体。我们的实验室专注于揭示MUTYH和MUTYH变种的基本特征。我们的基本假设是，对OG的详细分子理解：错配识别与揭示MUTYH与结直肠癌之间复杂的关系是齐头并进的。我们最近开发了哺乳动物细胞分析，我们将使用它来评估细胞中修饰底物的修复。这将使我们能够衡量催化和结合缺陷如何影响细胞的整体修复。我们最近对细菌酶的研究结果表明，OG的2-氨基对OG的初始定位至关重要，我们将通过制造额外的衍生物并使用单分子技术直接监控搜索过程来进一步验证这一假设。这将为初始损坏检测步骤中涉及的特征提供重要信息，并允许更好地预测进行损害检测的特征。我们还将结合我们的酶分析和细胞分析来深入了解区域间连接器(IDC)区域和我们最近发现的锌(II)协调位点在介导有效修复以及与下游修复蛋白和参与损伤信号传递的蛋白质之间的协调方面所起的作用。最后，我们还将详细说明MUTYH活性在增加细胞对烷基化损伤的敏感性方面所做的工作。作为这项工作的一部分，我们将 开发新的方法来标记细胞中MutY糖基酶活性的位置。这项工作的意义在于，它将提供关于MUTYH功能障碍的重要信息，无论是遗传的还是后天的，可能会为癌症的发生奠定基础。此外，MUTYH在介导细胞对氧化损伤和烷基化反应中的作用可能为靶向癌细胞提供新的策略。

 DESCRIPTION (provided by applicant): The MUTYH glycosylase has garnered the spotlight with the discovery of a correlation between inherited MUTYH variations and colorectal cancer (CRC). Using pre-steady kinetics and noncleavable 2'- deoxyadenosine analogue-containing oligonucleotides, we provided information on the functional defects of two missense variants of MUTYH found in colorectal cancer that was key for establishing the connection between MUTYH variants and colorectal cancer (now referred to a MUTYH-associated polyposis or MAP). MUTYH prevents mutations associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) by removal of misincorporated adenine residues from OG:A mismatches, and thus aberrant functioning MUTYH leads to the accumulation of mutations in tumor suppressor genes, like APC. Since the original discovery of MAP, over 100 different variants of MUTYH have been discovered. Our laboratory has focused on revealing fundamental features of MUTYH and MUTYH variants. Our underlying hypothesis is that a detailed molecular understanding of OG:A mismatch recognition goes hand-in-hand with revealing intricacies of the relationship between MUTYH and colorectal cancer. We have recently developed mammalian cell assays that we will use to evaluate the repair of modified substrates in cells. This will allow us to gauge how catalytic and binding defects impact overall repair in cells. We have recent results with the bacterial enzymes that suggests that the 2-amino- group of OG is critical for initial localization f OG, and we will further test this hypothesis by making additional derivatives and directly monitoring the search process using single-molecule techniques. This will provide important information into features that are involved in the initial damage detection step, and allow for better prediction of features that made compromise damage detection. We also will use our enzymatic assays in conjunction with cellular assays to provide insight into the role of the interdomain connector (IDC) region, and the Zn(II) coordination site we recently discovered on mediating efficient repair, and coordinating with downstream repair proteins, and proteins involved in damage signaling. Lastly, we also will elaborate on work that implicated MUTYH activity in increasing the sensitivity of cells to alkylation damage. As part of this work, we will develop novel methods to tag sites of MutY glycosylase activity in cells. The significance of this work is that it will provide important information as to the how MUTYH dysfunction, either inherited or acquired, may set the stage for carcinogenesis. In addition, the role of MUTYH in mediating cellular responses to oxidative damage and alkylation may provide new strategies to target cancer cells.