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DNA碱基损伤是最常见的细胞毒性和突变DNA损伤之一，它是 经典的由病变特异的DNA糖基酶修复，它切除烷基化的碱基以创建 并启动碱基切除修复(BER)途径。DNA烷基化修复对 基因组的稳定性，而且是癌症化疗的主要耐药因素，所以其他 研究较少，但生物学上关键的烷基化修复途径值得描述。因此，这项建议 重点介绍了重要的非糖基酶途径，通过这种途径，烷基化损伤可以通过直接 反转(目标1)，或通过非经典地将损伤引导到 主要的DNA切除修复途径(目标2-4)，以避免有毒DNA物种的释放。我们的努力是 Date已经帮助阐明了人直接逆转蛋白AGT(O6- 烷基鸟嘌呤-DNA-烷基转移酶)和ABH3(脱烷基双加氧酶AlkB同系物3)和 支持目标1中提出的它们的进一步特征。此外，我们还发现了三个系统 表征串扰，这是烷基化修复途径相交的重要细胞策略 促进受损DNA的非经典进入切除修复途径。因此，我们将 此外，还鉴定了三种特定的烷基化碱基损伤反应蛋白，它们促进非 经典进入DNA切除修复的三个原型途径：目标2)ATL(烷基- 转移酶不活跃但基因上与核苷酸切除修复相关的 (NER)，它切除扭曲DNA的巨大病变，目标3)AGTdoV(O6-烷基鸟嘌呤-DNA- 烷基转移酶-核酸内切酶)，将AGT与Endo V DNA骨架共价连接 切除酶以形成断裂，作为误码率的底物，并目的是糖基酶失活的MAG2 (甲基腺嘌呤糖基酶同系物2)，在基因和结构上与错配修复有关 (MMR)，它经典地切除不匹配的区域。我们建议将定量生物物理 用大分子X射线结晶学(MX)和Small表征蛋白质及其复合体 Tainer实验室溶液中的角X射线散射(SAXS)及其在体外和体内的补充细节 来自佩格实验室的活体生化和突变结果。拟议的工作将以核心为特征 烷基化修复起始蛋白及其体内结构功能研究 非糖基酶烷基化损伤修复关键环节的机制。总体而言，这些结果将 对与遗传完整性相关的烷基化损伤反应提供统一的理解 化疗耐药性，以及促进用于癌症治疗的烷基化抑制剂的进展。 因此，所获得的结果将有助于阐明dna烷基化修复蛋白、其抑制剂和步骤。 与新的治疗策略和癌症化疗相关。DNA烷基化是导致癌症易感性的基因组不稳定的一个来源，也是一个主要的 癌症化疗的结果。烷基化损伤可以通过反转碱基损伤或 通过招募非经典的修复机器来纠正损伤；然而，无论是结构化学 也不能完全理解这些通路所介导的“串扰”机制。我们建议 描述烷基化损伤修复的这两个关键方面的结构细胞生物学，这两个方面直接 与改进的癌症化疗和环境制剂的风险评估相关。

项目成果

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

烷基转移酶样蛋白：DNA修复途径之间的分子开关。

• 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.

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.

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