Structural Biochemistry of DNA Dealkylation

DNA 脱烷基化的结构生物化学

• 批准号: 8671412
• 负责人: John A. Tainer
• 金额: $ 3.5万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-17 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8671412
• 关键词: Alkylation; Bacteria; Base Excision Repairs; Binding; Biochemical; Biochemistry; Catalysis; Cellular biology; Chemotherapy-Oncologic Procedure; Complement; Complex; DNA; DNA Alkylation; DNA Damage; DNA glycosylase; DNA lesion; DNA-Directed RNA Polymerase; Data; Dealkylation; Detection; Development; Dioxygenases; Endonuclease V; Enzymes; Excision; Excision Repair; Genetic; Genetic Screening; Genome Stability; Genomic Instability; Homologous Gene; Human; In Vitro; Lead; Lesion; Light; Malignant Neoplasms; Mediating; Methods; Mismatch Repair; Molecular; Multiprotein Complexes; Nucleotide Excision Repair; O(6)-Methylguanine-DNA Methyltransferase; Pathway interactions; Predisposition; Proteins; Resistance; Risk Assessment; Roentgen Rays; Site; Solutions; Source; Specificity; Structural Biochemistry; Structural Chemistry; Structure; System; Techniques; Testing; Transferase; Translating; Vertebral column; Work; X-Ray Crystallography; Yeasts; adenine glycosylase; alkyltransferase; base; biophysical properties; cancer risk; cancer therapy; chemotherapy; comparative; cytotoxic; endo V; endonucleaseV; environmental agent; human DNA; improved; in vivo; inhibitor/antagonist; interdisciplinary approach; microbial; novel; novel therapeutics; protein complex; repaired; research study; resistance factors; response

Alkylated DNA base damage, one of the most common cytotoxic and mutagenic DNA lesions, is classically repaired by lesion-specific DNA glycosylases, which excise alkylated bases to create abasic sites and initiate the base-excision repair (BER) pathway. DNA alkylation repair is critical for genome stability and furthermore a major resistance factor for cancer chemotherapies, so the other less studied but biologically key alkylation repair pathways merit characterization. This proposal thus focuses upon important non-glycosylase pathways, whereby alkylation damage is removed by direct reversal (Aim 1), or by pathway `crosstalk' proteins that non-classically guide damage into one of the major DNA-excision repair pathways (Aims 2-4) to avoid release of toxic DNA species. Our efforts to date have helped elucidate the structural chemistry for human direct reversal proteins AGT (O6- alkylguanine-DNA-alkyltransferases) and ABH3 (the dealkylation dioxygenase AlkB homolog 3) and support their further characterizations proposed in Aim 1. We moreover discovered three systems to characterize crosstalk, an important cellular strategy for alkylation repair pathway intersection that promotes the non-classical entry of damaged DNA into excision repair pathways. We will therefore furthermore characterize three specific alkylation base damage response proteins that promote non- classical entry into each of the three prototypic pathways for DNA excision repair: Aim 2) ATL (alkyl- transferase-like) that is transferase-inactive but genetically connected to nucleotide excision repair (NER), which excises bulky lesions that distort DNA, Aim 3) AGTendoV (O6-alkylguanine-DNA- alkyltransferase-endonucleaseV) that covalently connects AGT with the Endo V DNA backbone excision enzyme to form breaks that are substrates for BER, and Aim 4) glycosylase-inactive Mag2 (methyl-adenine-glycosylase homolog 2) that genetically and structurally connects to mismatch repair (MMR) that classically excises mismatched regions. We propose to integrate quantitative biophysical characterization of proteins and complexes by macromolecular X-ray crystallography (MX) and small angle X-ray scattering in solution (SAXS) in the Tainer lab with complementary detailed in vitro and in vivo biochemical and mutational results from the Pegg lab. The proposed work will characterize core alkylation repair initiation proteins and their in vivo functions to elucidate structure-function mechanisms for key facets of non-glycosylase alkylation damage repair. Overall, these results will provide a unified understanding of alkylation damage responses relevant to genetic integrity, to chemotherapy resistance, and to promoting advances in alkylation inhibitors for cancer therapies. Results obtained will therefore shed light on DNA alkylation repair proteins, their inhibitors, and steps relevant to novel therapeutic strategies and cancer chemotherapies. DNA alkylation is a source of genomic instability leading to cancer predispositions, and is also a major result of cancer chemotherapies. Alkylation damage can be removed directly by reversing the base damage or by the recruitment of non-classical repair machinery to correct the lesion; yet, neither the structural chemistries nor the mechanisms of `crosstalk' mediated by these pathways are fully understood. We propose to characterize the structural cell biology of these two key facets of alkylation damage repair, which are directly relevant to improved cancer chemotherapies and risk assessments for environmental agents.

烷基化的DNA碱损伤是最常见的细胞毒性和诱变DNA病变之一，是 通过病变特异性的DNA糖基酶进行经典修复，该糖基酶切除烷基化碱以创建 abasic站点并启动基本拆卸维修（BER）途径。 DNA烷基化修复至关重要 基因组稳定性，以及癌症化学疗法的主要抗性因子，因此 研究较少但在生物学上关键的烷基化修复途径值得表征。因此，这一建议 专注于重要的非糖基化酶途径，该途径通过直接去除烷基化损伤 反转（AIM 1），或通过途径“串扰”蛋白，非经典损害损害 主要的DNA-拆卸修复途径（目标2-4），以避免释放有毒的DNA物种。我们的努力 日期有助于阐明人类直接逆转蛋白AGT的结构化学（O6-- 烷基鸟嘌呤-DNA-烷基转移酶）和ABH3（DealKylation dioxygenase AlkB同源物3）和 支持AIM 1中提出的进一步特征。我们还发现了三个系统 表征Crosstalk，这是烷基化修复途径相交的重要细胞策略， 促进受损DNA进入切除修复途径的非古典入口。因此，我们会 此外，表征了三种特定的烷基化碱损伤反应蛋白，这些蛋白会促进非 - 经典进入三种原型途径的DNA切除修复：目标2）ATL（烷基 - 类似转移酶）是转移酶不活性但遗传上连接到核苷酸切除修复 （NER），造成DNA扭曲的笨重病变，AIM 3）Agtendov（O6-烷基Guanine-DNA- 烷基转移酶 - 核酸固定酶，它与Endo V DNA骨架共价连接 切除酶形成BER的底物的断裂，AIM 4）糖基酶无效MAG2 （甲基 - 腺苷 - 糖基化酶同源2）在遗传和结构上连接不匹配修复 （MMR）经典消除不匹配的区域。我们建议整合定量生物物理 大分子X射线晶体学（MX）和小的蛋白质和复合物表征 泰纳实验室中的溶液中的角度X射线散射（SAX），其体外和互补的详细信息 Pegg Lab的体内生化和突变结果。拟议的工作将表征核心 烷基化修复起始蛋白及其体内功能以阐明结构功能 非糖基化酶烷基化损伤修复的关键方面的机制。总的来说，这些结果将 提供与遗传完整性相关的烷基化损伤反应的统一理解 化学疗法耐药性，并促进癌症疗法烷基化抑制剂的进步。 因此，获得的结果将阐明DNA烷基化修复蛋白，其抑制剂和步骤 与新颖的治疗策略和癌症化学疗法有关。 DNA烷基化是导致癌症易感性的基因组不稳定性的来源，也是主要的 癌症化学疗法的结果。烷基化损伤可以通过逆转基本损坏或 通过招募非古典修复机制来纠正病变；但是，结构化学都不是 这些途径介导的“串扰”的机制也没有完全理解。我们建议 表征这两个关键方面的烷基化损伤修复的结构细胞生物学，直接 与改善环境药物的癌症化学疗法和风险评估有关。

项目成果

Alkyltransferase-like proteins: molecular switches between DNA repair pathways.

• DOI: 10.1007/s00018-010-0405-8
• 发表时间: 2010-11
• 期刊: CELLULAR AND MOLECULAR LIFE SCIENCES
• 影响因子: 8
• 作者: Tubbs, Julie L.;Tainer, John A.
• 通讯作者: Tainer, John A.

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation-π interactions.

烷基转移酶样蛋白 (Atl1) 通过阳离子-β 相互作用区分用于 DNA 修复的烷基化鸟嘌呤。

• DOI: 10.1073/pnas.1209451109
• 发表时间: 2012
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Wilkinson,OliverJ;Latypov,Vitaly;Tubbs,JulieL;Millington,ChristopherL;Morita,Rihito;Blackburn,Hannah;Marriott,Andrew;McGown,Gail;Thorncroft,Mary;Watson,AmandaJ;Connolly,BernardA;Grasby,JaneA;Masui,Ryoji;Hunter,Christopher
• 通讯作者: Hunter,Christopher

Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines.

• DOI: 10.1016/j.molcel.2012.04.028
• 发表时间: 2012-07-13
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Latypov, Vitaly F.;Tubbs, Julie L.;Watson, Amanda J.;Marriott, Andrew S.;McGown, Gail;Thorncroft, Mary;Wilkinson, Oliver J.;Senthong, Pattama;Butt, Amna;Arvai, Andrew S.;Millington, Christopher L.;Povey, Andrew C.;Williams, David M.;Santibanez-Koref, Mauro F.;Tainer, John A.;Margison, Geoffrey P.
• 通讯作者: Margison, Geoffrey P.