Mechanisms of selective excision and oxidative repair of alkylated DNA

烷基化DNA的选择性切除和氧化修复机制

• 批准号: 8306937
• 负责人: Manel Camps
• 金额: $ 33.9万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-25 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306937
• 关键词: Acute Erythroblastic Leukemia; Address; Adenine; Alkylating Agents; Alkylation; Antineoplastic Agents; Base Excision Repairs; Biochemical; Bone Marrow; Cancer Etiology; Catalysis; Cell Death; Cells; Chemotherapy-Oncologic Procedure; Chronic; Clinical; Complex; DNA; DNA Adducts; DNA Alkylation; DNA Damage; DNA Repair Enzymes; DNA Synthesis Inhibition; DNA biosynthesis; DNA glycosylase; Data; Dealkylation; Dioxygenases; Discrimination; Enzymes; Escherichia coli; Evolution; Excision; Exhibits; Exposure to; Family; Fission Yeast; Foundations; Genetic; Genome Stability; Genomic Instability; Goals; Health; Human; Individual; Inflammation; Inflammatory; Knowledge; Laboratories; Lead; Lesion; Malignant Neoplasms; Mammalian Cell; Metabolism; Methods; Methylation; Modeling; Modification; Molecular; Mus; Mutagens; Myelin Associated Glycoprotein; Normal Cell; Nucleotides; Oligonucleotides; Organism; Orthologous Gene; Pathway interactions; Patients; Phylogenetic Analysis; Play; Polymerase; Process; Reactive Oxygen Species; Resistance; Role; Specificity; Structure; Substrate Specificity; System; Testing; Therapeutic; Toxic Environmental Substances; Toxic effect; Work; Yeasts; adduct; base; cancer risk; cancer therapy; chemotherapy; comparative; cytotoxic; cytotoxicity; demethylation; design; directed evolution; human DNA; improved; inhibitor/antagonist; insight; mutant; novel; oxidant stress; pressure; repaired; small molecule; structural biology; therapy resistant; tissue/cell culture; tool; tumor

DESCRIPTION (provided by applicant): N3-methyladenine (3mA) and 1,N6-ethenoadenine (eA) are two DNA base modifications produced from exposure to environmental genotoxic agents, cellular metabolites, and anti-cancer drugs. 3mA lesions are highly cytotoxic owing to their inhibition of DNA synthesis by polymerases, and this cytotoxicity is a rationale for the use of alkylating agents in cancer chemotherapy. eA, which is associated with chronic inflammatory conditions, is highly mutagenic and can lead to genomic instability and cancer. Two different, partially redundant enzymatic activities have evolved for these two specific lesions: i) oxidative demethylation by DNA dioxygenases and ii) base excision repair by DNA glycosylases. The precise determinants for the specificity and catalysis of these enzymes toward 3mA and eA remain unclear. We seek to fill this critical gap in knowledge by a unique integration of directed evolution and structural biology methods in order to obtain a comprehensive mechanistic understanding of 3mA and eA selection and catalysis by human ABH2 dioxygenase (Aim 1) and the yeast family of MAG 3mA glycosylases (Aim 2). This work capitalizes on the convergent evolution observed between the two repair systems, and is based on our preliminary results that have identified ABH2 mutants with the capacity to protect cells from 3mA toxicity. We will test the hypothesis that ABH2 repair of 3mA, unlike that of other known substrates, involves excision and further processing by base excision repair. Our general approach for each aim is to i) identify residues important for substrate discrimination using directed evolution under selective alkylation pressure, ii) determine crystal structures of ABH2 and MAG proteins in complex with 3mA- and eA-DNA, and iii) test the contribution of individual residues toward 3mA and eA specificity and repair. These studies will provide novel insight into how these enzymes determine the fate of cytotoxic and mutagenic lesions toward a particular repair pathway. In addition, in Aim 1 we probe the translational implications of our ABH2 mutants for cancer treatment with methylating agents using a mouse erythroleukemia (MEL) cell tissue culture model. Our studies have at least three direct clinical implications. First, etheno-DNA adducts likely play a role in the etiology of cancer associated with chronic inflammation, and thus results on eA repair may provide new ways to determine the risk of cancer in patients suffering from chronic inflammatory conditions. Second, our 3mA-protecting ABH2 mutants have direct implications for understanding the origins of resistance to therapy with methylating agents in tumors and for the design of new chemotherapeutic approaches involving bone marrow protection. Third, our structure-function studies on 3mA glycosylase repair are a necessary first step for the design of small molecule inhibitors as a way to enhance the cytotoxicity of methylating agents.

描述（由申请人提供）：N3-甲基腺嘌呤（3 mA）和1，N6-乙烯基腺嘌呤（eA）是暴露于环境遗传毒性物质、细胞代谢物和抗癌药物后产生的两种DNA碱基修饰。3 mA损伤由于其通过聚合酶抑制DNA合成而具有高度细胞毒性，并且这种细胞毒性是在癌症化疗中使用烷化剂的基本原理。eA与慢性炎性疾病相关，具有高度致突变性，可导致基因组不稳定和癌症。对于这两种特异性病变，已经进化出两种不同的、部分冗余的酶活性：i）通过DNA双加氧酶的氧化去甲基化和ii）通过DNA糖基化酶的碱基切除修复。这些酶对3 mA和eA的特异性和催化作用的精确决定因素仍不清楚。我们试图填补这一关键的知识空白，通过独特的整合定向进化和结构生物学方法，以获得全面的机械理解3 mA和eA的选择和催化的人ABH 2双加氧酶（目标1）和酵母家族的MAG 3 mA糖基化酶（目标2）。这项工作利用了在两个修复系统之间观察到的趋同进化，并且基于我们的初步结果，这些结果已经鉴定出ABH 2突变体具有保护细胞免受3 mA毒性的能力。我们将测试的假设，ABH 2修复3 mA，不像其他已知的基板，涉及切除和进一步处理的碱基切除修复。我们对每个目标的一般方法是i）在选择性烷基化压力下使用定向进化鉴定对于底物区分重要的残基，ii）确定与3 mA-和eA-DNA复合的ABH 2和MAG蛋白的晶体结构，和iii）测试单个残基对3 mA和eA特异性和修复的贡献。这些研究将为这些酶如何决定细胞毒性和致突变性病变向特定修复途径的命运提供新的见解。此外，在目标1中，我们使用小鼠红白血病（MEL）细胞组织培养模型探讨了ABH 2突变体对甲基化剂治疗癌症的翻译影响。我们的研究至少有三个直接的临床意义。首先，乙烯基-DNA加合物可能在与慢性炎症相关的癌症病因学中发挥作用，因此eA修复的结果可能提供新的方法来确定患有慢性炎症性疾病的患者的癌症风险。第二，我们的3 mA保护ABH 2突变体有直接的影响，了解的起源与甲基化剂治疗肿瘤和设计新的化疗方法，涉及骨髓保护。第三，我们对3 mA糖基化酶修复的结构-功能研究是设计小分子抑制剂作为增强甲基化剂细胞毒性的一种方式的必要的第一步。