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诱变性 DNA 损伤之一 通常由损伤特异性 DNA 糖基化酶修复，该酶切除烷基化碱基以产生 无碱基位点并启动碱基切除修复（BER）途径。 DNA 烷基化修复对于 基因组稳定性，也是癌症化疗的主要耐药因素，因此其他 研究较少但生物学上关键的烷基化修复途径值得表征。因此该提案 专注于重要的非糖基化酶途径，通过直接消除烷基化损伤 逆转（目标 1），或通过途径“串扰”蛋白非经典地将损伤引导至其中之一 主要 DNA 切除修复途径（目标 2-4）以避免有毒 DNA 物质的释放。我们的努力 日期有助于阐明人类直接逆转蛋白 AGT（O6- 烷基鸟嘌呤-DNA-烷基转移酶）和 ABH3（脱烷基双加氧酶 AlkB 同源物 3）和 支持目标 1 中提出的进一步表征。我们还发现了三个系统 表征串扰，这是烷基化修复途径交叉的重要细胞策略 促进受损 DNA 非经典进入切除修复途径。因此我们将 进一步表征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 切除修复的三种原型途径中的每一种：目标 2) ATL（烷基- 转移酶样），转移酶无活性，但在基因上与核苷酸切除修复有关 (NER)，切除扭曲 DNA 的大块病变，目标 3) AGTendoV (O6-烷基鸟嘌呤-DNA- 烷基转移酶-核酸内切酶 V）将 AGT 与 Endo V DNA 主链共价连接 切除酶形成断裂，作为 BER 的底物，目标 4) 糖基化酶失活 Mag2 （甲基腺嘌呤糖基酶同源物 2）在遗传和结构上与错配修复相关 (MMR)，通常切除不匹配的区域。我们建议整合定量生物物理 通过大分子 X 射线晶体学 (MX) 和小分子 X 射线晶体学表征蛋白质和复合物 Tainer 实验室的溶液中角 X 射线散射 (SAXS)，具有体外和体内的补充详细信息 Pegg 实验室的体内生化和突变结果。拟议的工作将体现核心特征 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关键方面的机制。总体而言，这些结果将 提供对与遗传完整性相关的烷基化损伤反应的统一理解， 化疗耐药性，并促进癌症治疗烷基化抑制剂的进展。 因此，获得的结果将揭示 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根源，也是一个主要因素 癌症化疗的结果。烷基化损伤可以通过逆转基础损伤直接消除或 通过使用非经典修复机制来纠正病变；然而，结构化学 由这些途径介导的“串扰”机制也尚未完全了解。我们建议 表征烷基化损伤修复这两个关键方面的结构细胞生物学，它们直接 与改进癌症化疗和环境因素风险评估相关。

项目成果

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。

• 发表时间: 2012-11-13
• 影响因子: 11.1
• 作者: Wilkinson, Oliver J;Latypov, Vitaly;Tubbs, Julie L;Millington, Christopher L;Morita, Rihito;Blackburn, Hannah;Marriott, Andrew;McGown, Gail;Thorncroft, Mary;Watson, Amanda J;Connolly, Bernard A;Grasby, Jane A;Masui, Ryoji;Hunter, Christopher
• 通讯作者: Hunter, Christopher

P53 conformational switching for selectivity may reveal a general solution for specific DNA binding.

P53 构象转换的选择性可能揭示特定 DNA 结合的通用解决方案。

• 发表时间: 2011-06-01
• 作者: Tubbs, Julie L;Tainer, John A
• 通讯作者: Tainer, John A

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。

• 发表时间: 2010-11
• 作者: Tubbs, Julie L;Tainer, John A
• 通讯作者: Tainer, John A

Structural basis of O6-alkylguanine recognition by a bacterial alkyltransferase-like DNA repair protein.

细菌烷基转移酶样 DNA 修复蛋白识别 O6-烷基鸟嘌呤的结构基础。

• 发表时间: 2010-04-30
• 作者: Aramini, James M;Tubbs, Julie L;Kanugula, Sreenivas;Rossi, Paolo;Ertekin, Asli;Maglaqui, Melissa;Hamilton, Keith;Ciccosanti, Colleen T;Jiang, Mei;Xiao, Rong;Soong, Ta;Rost, Burkhard;Acton, Thomas B;Everett, John K;Pegg, Anthony E;Taine
• 通讯作者: Taine

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

Atl1 调节全局基因组和 O(6)-烷基鸟嘌呤转录偶联修复之间的选择。

• 发表时间: 2012-07-13
• 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
• 通讯作者: Santibanez