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: 8478102
• 负责人: Manel Camps
• 金额: $ 33.17万
• 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/8478102
• 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（3mA）和1,N6-乙烯腺嘌呤（eA）是由于暴露于环境基因毒性剂、细胞代谢物和抗癌药物而产生的两种DNA碱基修饰。 3mA 损伤由于聚合酶抑制 DNA 合成而具有高度细胞毒性，这种细胞毒性是在癌症化疗中使用烷化剂的基本原理。 eA 与慢性炎症相关，具有高度诱变性，可导致基因组不稳定和癌症。对于这两种特定损伤，已进化出两种不同的、部分冗余的酶活性：i) DN​​A 双加氧酶的氧化去甲基化和 ii) DN​​A 糖基化酶的碱基切除修复。这些酶对 3mA 和 eA 的特异性和催化作用的精确决定因素仍不清楚。我们寻求通过定向进化和结构生物学方法的独特整合来填补这一关键知识空白，以获得对人类 ABH2 双加氧酶（目标 1）和 MAG 3mA 糖基酶酵母家族（目标 2）的 3mA 和 eA 选择和催化作用的全面机制理解。这项工作利用了两个修复系统之间观察到的趋同进化，并基于我们的初步结果，该结果已鉴定出具有保护细胞免受 3mA 毒性能力的 ABH2 突变体。我们将测试以下假设：3mA 的 ABH2 修复与其他已知底物的修复不同，涉及切除和通过碱基切除修复进行进一步处理。我们针对每个目标的一般方法是 i) 使用选择性烷基化压力下的定向进化来识别对底物区分重要的残基，ii) 确定与 3mA-和 eA-DNA 复合的 ABH2 和 MAG 蛋白的晶体结构，以及 iii) 测试单个残基对 3mA 和 eA 特异性和修复的贡献。这些研究将为这些酶如何决定特定修复途径的细胞毒性和诱变损伤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 ABH2 突变体对甲基化药物癌症治疗的翻译影响。我们的研究至少具有三个直接的临床意义。首先，乙烯-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 修复结果可能为确定患有慢性炎症的患者患癌症的风险提供新方法。其次，我们的 3mA 保护性 ABH2 突变体对于了解肿瘤中甲基化药物治疗耐药的起源以及设计涉及骨髓保护的新化疗方法具有直接意义。第三，我们对 3mA 糖基化酶修复的结构功能研究是设计小分子抑制剂以增强甲基化剂细胞毒性的必要的第一步。