Structural Biochemistry of DNA Dealkylation

DNA 脱烷基化的结构生物化学

• 批准号: 8081653
• 负责人: John A. Tainer
• 金额: $ 12.06万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8081653
• 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; Figs - dietary; 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; 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; public health relevance; repaired; research study; resistance factors; response

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE 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.

描述（由申请人提供）：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糖基酶经典修复的，该糖基化酶会切除烷基化碱，以创建无碱性位点并启动碱基化修复（BER）修复（BER）。 DNA烷基化修复对于基因组稳定性至关重要，而且是癌症化学疗法的主要抗药性因子，因此其他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，该提案的重点是重要的非糖基化酶途径，从而通过直接逆转（AIM 1）或通过途径“串扰”蛋白去除烷基化损伤，该蛋白非经典地指导损害损害主要的DNA分离修复途径之一（AIMS 2-4），以避免释放毒性DNA。我们迄今为止的努力有助于阐明人类直接逆转蛋白AGT的结构化学（O6-烷基瓜氨基-DNA-烷基转移酶）和ABH3（交易二氧酶AlkB同源物3），并支持其在AIM中提出的进一步特征1。损坏的DNA进入切除修复途径。 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-烷基鸟嘌呤-DNA-烷基转移酶 - 内核酸），将AGT与Endo v DNA骨架骨架切除酶连接起来，形成是BER底物的断裂，并且AIM 4）糖基质酶无效的MAG2无关（甲基 - 添合添加剂）与甲基 - 添合糖基酶构造的（属糖基酶2），并将其连接起来，并将其连接起来。经典切除不匹配的区域。我们建议通过大分子X射线晶体学（MX）（MX）和Tainer Lab中溶液中的小角度X射线散射对蛋白质和复合物进行定量生物物理特性，并具有互补的体外和体内生物化学和PEGG LAB的突变结果。提出的工作将表征核心烷基化修复起始蛋白及其体内功能，以阐明非糖基化酶烷基化损伤修复的关键方面的结构功能机制。总体而言，这些结果将提供对烷基化损伤反应的统一理解，与遗传完整性相关，与化学疗法抗性以及促进癌症治疗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，其抑制剂以及与新型治疗策略和癌症化学疗法有关的步骤。公共卫生相关性DNA烷基化是导致癌症易感性的基因组不稳定性的来源，也是癌症化学疗法的主要结果。可以通过逆转基本损伤或募集非经典修复机制来纠正病变来直接消除烷基化损伤；然而，这些途径介导的结构化学和“串扰”的机制均未完全理解。我们建议表征烷基化损伤修复的这两个关键方面的结构细胞生物学，这与改善的癌症化学疗法和环境药物的风险评估直接相关。