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

MUTYH 和 MUTYH 变体与结直肠癌相关的 DNA 损伤修复

• 批准号: 8041452
• 负责人: SHEILA Sue DAVID
• 金额: $ 21.86万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8041452
• 关键词: 2' -deoxyadenosine; 8-Oxo-2' -Deoxyguanosine; Adenine; Affect; Affinity; Baculoviruses; Base Excision Repairs; Base Pairing; Binding; Biological Assay; Biology; Cell Line; Cells; Cellular Assay; Chemicals; Clinical Data; Colorectal Cancer; Complement; Complex; DNA Damage; DNA Repair; DNA Repair Enzymes; Defect; Enzymatic Biochemistry; Enzymes; Escherichia coli; Etiology; Excision; Functional disorder; Gene Mutation; Genomics; Goals; Homologous Gene; Human; Inherited; Insecta; Kinetics; Laboratories; Lead; Location; Mammalian Cell; Measures; Mediating; Molecular; Mutate; Mutation; Nucleic Acids; Oligonucleotides; Oxidative Stress; Patients; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Predisposition; Prevention; Process; Property; Proteins; Research; Role; Staging; Syndrome; Variant; Work; adenine glycosylase; analog; base; insight; mimetics; polyposis; prevent; programs; repaired; research study; response; tumorigenesis

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. PUBLIC HEALTH RELEVANCE: This project will provide information on the functional consequences of variations in the DNA repair enzyme MUTYH that are correlated with the inherited colorectal cancer syndrome known as MUTYH-associated polyposis (MAP). This information will provide insight into how MUTYH variations lead to dysfunction in DNA repair resulting in the accumulation of deleterious mutations, and thereby setting the stage for colorectal cancer.

描述（由申请人提供）：大肠杆菌MutY及其人类同源物（MUTYH，前身为MYH）通过去除OG:A错配中的错配腺嘌呤残基，在预防8-氧-7,8-二氢-2'-脱氧鸟苷（OG）相关突变中发挥重要作用。由于遗传MUTYH变异与结直肠癌（CRC）之间的相关性的发现，这种糖基化酶引起了人们的关注。利用前稳态动力学和不可切割的含2'-脱氧腺苷类似物的寡核苷酸，我们提供了在结直肠癌中发现的两种MUTYH错义变体的功能缺陷的信息，这是建立MUTYH变体与结直肠癌之间联系的关键（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。临床数据正在迅速涌现，MUTYH的新突变也在不断被发现。然而，目前尚不清楚是否所有的MUTYH变异都会影响腺嘌呤糖基酶活性，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与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酶的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与结直肠癌之间复杂的关系密切相关。具体来说，我们将开发细胞分析来揭示影响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，以评估大肠杆菌和哺乳动物细胞系中MutY和MUTYH错配的OG:A修复。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中特定的催化和结合缺陷来衡量这些基于细胞的检测的反应。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检验翻译后和与蛋白质伴侣相互作用对MUTYH活性的重要性。杆状病毒感染的昆虫细胞中表达的MUTYH被磷酸化，我们将确定这些磷酸化位点在MUTYH中的位置。我们将在MUTYH中制备相关的模拟磷酸化和消融磷酸化突变，并分析突变酶的腺嘌呤糖基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变异对内在酶特性、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酶活性、错配亲和力、在细菌和哺乳动物细胞试验中介导OG:A修复的能力、表达MUTYH变异的细胞的基因组突变率和对氧化应激的敏感性。这些信息将提供关于MUTYH变体是否功能失调以及功能失调的来源的信息。