DNA damage repair by MUTYH and MUTYH variants associated with colorectal cancer

描述（由申请人提供）：E。大肠杆菌MutY及其人类同源物（MUTYH，以前称为MYH）通过从OG：A错配中去除错误掺入的腺嘌呤残基，在预防与8-氧代-7，8-二氢-2 '-脱氧鸟苷（OG）相关的突变中起重要作用。这种糖基化酶因发现遗传性MUTYH变异与结直肠癌（CRC）之间的相关性而备受关注。使用预稳态动力学和含有不可切割的2 '-脱氧腺苷类似物的寡核苷酸，我们提供了关于在结直肠癌中发现的MUTYH的两个错义变体的功能缺陷的信息，这是建立MUTYH变体和结直肠癌之间的联系的关键（称为MUTYH相关息肉病或MAP）。对这种易感机制的理解仍处于早期阶段。临床数据正在迅速出现，MUTYH的新突变不断被发现。然而，在这一点上，尚不清楚是否所有的MUTYH变异都影响腺嘌呤糖基化酶活性，或者由于其他原因导致OG：A修复减少。我们建议进一步了解MUTYH和结直肠癌之间的关系，并继续了解这种独特的碱基切除修复糖基化酶错配识别的复杂特征。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和结直肠癌之间关系的复杂性密切相关。具体来说，我们将开发细胞测定来揭示影响MUTYH识别和修复OG：A错配的特征。这将涉及开发几种检测方法来评估大肠杆菌中MutY和MUTYH的OG：A错配的修复。大肠杆菌和哺乳动物细胞系。我们还将研究受损底物的改变对MUTYH介导的修复的影响。此外，我们将通过评估MUTYH中的特异性催化和结合缺陷来衡量这些基于细胞的测定的响应。我们还将使用腺嘌呤糖基化酶测定、错配结合实验和细胞修复测定来研究翻译后和与蛋白质伴侣的相互作用对MUTYH活性的重要性。在杆状病毒感染的昆虫细胞中表达的MUTYH被磷酸化，我们将鉴定MUTYH中这些磷酸化位点的位置。我们将在MUTYH中制备相关的磷酸模拟和磷酸消融突变，并分析突变酶的腺嘌呤糖基化酶活性和错配结合亲和力。我们将确定在结直肠癌中发现的MUTYH变异对固有酶性质、介导OG：A修复和防止细胞突变的能力的影响。这将包括测量腺嘌呤糖基化酶活性、错配亲和力、在细菌和哺乳动物细胞测定中介导OG：A修复的能力、基因组突变率和表达MUTYH变体的细胞对氧化应激的敏感性。该信息将提供关于变体MUTYH是否功能障碍以及功能障碍的起源的信息。

DESCRIPTION (provided by applicant): E. coli MutY and its human homologue (MUTYH, formerly MYH) play an important role in the prevention of mutations associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) by removal of misincorporated adenine residues from OG:A mismatches. This glycosylase has garnered the spotlight by 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 (referred to a MUTYH-associated polyposis or MAP). The understanding of this predisposition mechanism is still at an early stage. The clinical data is emerging rapidly, and new mutations in MUTYH continue to be uncovered. However, at this point, it is not clear whether all of the MUTYH variations affect the adenine glycosylase activity, or result in reduced OG:A repair for other reasons. We propose to further our understanding of the relationship between MUTYH and colorectal cancer, as well as continue our understanding of the complex features of mismatch recognition by this unique base-excision repair glycosylase. 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. Specifically, we will develop cellular assays to reveal features that affect recognition and repair of OG:A mismatches by MUTYH. This will involve developing several assays to assess the repair of OG:A mismatches of MutY and MUTYH in E. coli and mammalian cell lines. We will also examine the effects of alterations of the damaged substrate on MUTYH-mediated repair. In addition, we will gauge the response of these cell based assays by evaluating specific catalytic and binding defects in MUTYH. We will also examine the importance of post-translational and interactions with protein partners on the activity of MUTYH using adenine glycosylase assays, mismatch binding experiments and cellular repair assays. MUTYH expressed in baculovirus-infected insect cells is phosphorylated and we will identify the locations of these phosphorylation sites in MUTYH. We will prepare the relevent phospho-mimetic and phospho-ablating mutations in MUTYH and analyze the adenine glycosylase activity and mismatch binding affinity of the mutated enzymes We will determine the consequences of MUTYH variations found in colorectal cancer on the instrinsic enzymatic properties, ability to mediate OG:A repair and prevent mutations in cells. This will include measuring the adenine glycosylase activity, mismatch affinity, the ability to mediate OG:A repair in both bacterial and mammalian cellular assays, the genomic mutation rate and sensitivity of cells expressing MUTYH variants to oxidative stress. This information will provide information as to whether a variant MUTYH is dysfunctional, and the origin of the dysfunction.