Structural Biochemistry of DNA Dealkylation

描述（由申请人提供）：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，通常通过病变特异性DNA糖基酶修复，该酶切除烷基化碱基以产生基本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关键的烷基化修复途径值得表征。因此，该建议侧重于重要的非糖基化酶途径，通过直接逆转（目的1）或通过途径“串扰”蛋白将烷基化损伤非经典地引导损伤进入主要DNA切除修复途径之一（目的2-4），以避免释放有毒的DNA物种。迄今为止，我们的努力已经帮助阐明了人类直接逆转蛋白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）的结构化学，并支持了Aim 1中提出的进一步表征。此外，我们还发现了三个表征串扰的系统，串扰是烷基化修复通路交叉的重要细胞策略，可促进受损DNA进入切除修复通路的非经典进入。因此，我们将进一步表征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：目标2)ATL（烷基转移酶样），它不具有转移酶活性，但基因上与核苷酸切除修复（NER）相关，它可以切除扭曲DNA的大块病变；目标3)AGTendoV (o6 -烷基鸟嘌呤-DNA-烷基转移酶内切酶ev)，它将AGT与Endo V DNA主干切除酶共价连接，形成断裂，作为BER的底物；和Aim 4)糖基酶失活的Mag2（甲基腺嘌呤糖基酶同源物2），它在遗传和结构上与错配修复（MMR）联系在一起，后者通常会切除错配区域。我们建议将Tainer实验室的大分子x射线晶体学（MX）和溶液中小角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实验室的补充详细的体外和体内生化和突变结果结合起来。这项工作将描述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关键方面的结构-功能机制。总的来说，这些结果将提供与遗传完整性、化疗耐药性相关的烷基化损伤反应的统一理解，并促进用于癌症治疗的烷基化抑制剂的进展。因此，获得的结果将阐明DNA烷基化修复蛋白，它们的抑制剂，以及与新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的一个来源，也是癌症化疗的主要结果。烷基化损伤可直接通过逆转碱基损伤或通过招募非经典修复机械来纠正病变来消除；然而，结构化学和由这些途径介导的“串扰”机制都没有被完全理解。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特征，这与改进癌症化疗和环境因子风险评估直接相关。

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.