DNA Damage Repair by MUTYH and MUTYH Variants Associated with Colorectal Cancer

MUTYH相关息肉病（MAP）是一种遗传性结直肠癌（CRC）综合征， MUTYH碱基切除修复（BER）糖基化酶基因中的遗传性双等位基因突变。本实验室 提供了在结肠直肠癌中发现的MUTYH的两个错义变体的功能缺陷的酶分析。 这是建立MUTYH变异体与结直肠癌之间联系的关键。MUTYH 通过去除错误掺入的 来自OG的腺嘌呤残基：A错配，因此功能异常的MUTYH导致 肿瘤抑制基因的突变。自从最初发现MAP以来， 在MUTYH报道。本项目的重点是揭示MUTYH和MUTYH的基本特征 变体。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和癌症之间错综复杂的关系密切相关。通过关联 结果来自酶测定和E.大肠杆菌和哺乳动物细胞， 损伤识别和碱基切除中特异性分子缺陷对MUTYH介导的整体 修复.这导致发现了许多新的令人惊讶的MUTYH介导的修复特征，这些特征将成为MUTYH介导修复的基础。 这是这次更新申请的重点。具体来说，我们将使用涉及核酸的多方面方法， 化学、酶学、单分子实验和细胞分析，以分析大量MUTYH 位于两个金属辅因子和FSH OG识别基序附近的变体，揭示了对关键的 影响损伤识别和修复的特征。我们还将评估MUTYH变异体在 染色质的背景。我们还将评估MUTYH在染色质中的损伤识别可能是如何进行的。 由损伤传感器UV-DDB协助。我们最近发现MUTYH与DNA中的脱碱基位点结合， 介导对烷基化损伤的细胞毒性反应。我们将确定MUTYH与 无碱基位点是MUTYH参与DNA应答的潜在起源 OG以外的损伤：A错配，如紫外线和化疗DNA损伤剂。的 这项工作的意义在于，它将为MUTYH的机制提供重要信息。 功能障碍，无论是遗传性的还是获得性的，都为癌症的发生奠定了基础，这些信息将直接 影响适当的患者管理。此外，MUTYH和MUTYH变体在介导细胞凋亡中的作用也被证实。 细胞对氧化损伤、烷基化和紫外线的反应可能为创新性的新方法提供基础。 治疗方法。

MUTYH-associated polyposis (MAP) is an inherited colorectal cancer (CRC) syndrome that results from inherited biallelic mutations in the gene for the MUTYH base excision repair (BER) glycosylase. Our laboratory provided enzymatic analysis 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. 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 aberrantly functioning MUTYH leads to the accumulation of mutations in tumor suppressor genes. Since the original discovery of MAP over 300 different mutations have been reported in MUTYH. This project is 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 cancer. By corrrelating results from enzymatic assays with cellular assays in E. coli and mammalian cells we have uncovered the impact of specific molecular defects in damage recognition and base excision on overall MUTYH-mediated repair. This has led to uncovering many new surprising features of MUTYH-mediated repair that will be the focus of this renewal grant application. Specifically, we will use a multi-faceted approach involving nucleic acid chemistry, enzymology, single-molecule experiments and cellular assays to analyze a large number of MUTYH variants localized near the two metal cofactors and FSH OG recognition motif to reveal insight into critical features impacting damage recognition and repair. We also will evaluate the impact of MUTYH variants in the context of chromatin. We will also evaluate how damage recognition by MUTYH in chromatin may be facilitated by the damage sensor UV-DDB. We recently showed that MUTYH binding to abasic sites in DNA mediates a cytotoxic response to alkylation damage. We will determine if the interaction of MUTYH with abasic sites is the underlying origin of the growing implication of MUTYH involvement in the response to DNA damage beyond OG:A mispairs, such as UV light and chemotherapeutic DNA damaging agents. The significance of this work is that it will provide important information into the mechanisms by which MUTYH dysfunction, either inherited or acquired, sets the stage for carcinogenesis and this information will directly influence appropriate patient management. In addition, the role of MUTYH and MUTYH variants in mediating celllular responses to oxidative damage, alkylation and UV light may provide the basis for innovative new therapeutic approaches.