Structural Biochemistry of DNA Dealkylation

描述（由申请方提供）：烷基化DNA碱基损伤是最常见的细胞毒性和致突变性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脱碱基位点并启动碱基切除修复（BER）途径。DNA烷基化修复对基因组稳定性至关重要，而且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，该建议集中在重要的非糖基化酶途径上，由此通过直接逆转（Aim 1）或通过途径“串扰”蛋白去除烷基化损伤，所述途径“串扰”蛋白非经典地引导损伤进入主要DNA切除修复途径之一（Aim 2-4）以避免释放有毒DNA物质。我们迄今的努力有助于阐明人类直接逆转蛋白AGT（O 6-烷基鸟嘌呤-DNA-烷基转移酶）和ABH 3（脱烷基化双加氧酶AlkB同系物3）的结构化学，并支持他们在目标1中提出的进一步表征。此外，我们发现了三个系统来表征串扰，一个重要的细胞策略，烷基化修复途径的交叉，促进非经典的损伤DNA进入切除修复途径。因此，我们将进一步表征三种特异性烷基化碱基损伤反应蛋白，其促进非经典进入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），其是转移酶失活的，但与核苷酸切除修复（NER）遗传相关，其切除扭曲DNA的大块病变，3）AGTendoV（O 6-烷基鸟嘌呤-DNA-烷基转移酶-内切核酸酶V），其将AGT与Endo V DNA骨架切除酶共价连接以形成作为BER底物的断裂，和目的4）糖基化酶失活的Mag 2（甲基-腺嘌呤-糖基化酶同源物2），其在遗传上和结构上与错配修复（MMR）连接，所述错配修复（MMR）典型地切除错配区域。我们建议将Tainer实验室中的大分子X射线晶体学（MX）和溶液中的小角X射线散射（SAXS）对蛋白质和复合物的定量生物物理表征与Pegg实验室的体外和体内生物化学和突变结果进行补充。拟议的工作将表征核心烷基化修复起始蛋白及其在体内的功能，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，这些结果将提供一个统一的认识烷基化损伤反应相关的遗传完整性，化疗耐药性，并促进烷基化抑制剂的癌症治疗的进展。因此，所获得的结果将揭示DNA烷基化修复蛋白，其抑制剂，以及与新的治疗策略和癌症化疗相关的步骤。DNA烷基化是导致癌症易感性的基因组不稳定性的来源，也是癌症化疗的主要结果。烷基化损伤可以通过逆转碱基损伤或通过招募非经典修复机制来纠正损伤而直接去除;然而，这些途径介导的结构化学和“串扰”机制都没有完全理解。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，这与改善癌症化疗和环境因子的风险评估直接相关。

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.