Structural Biochemistry of DNA Dealkylation

描述(申请人提供)：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，经典的修复方法是病变特定的DNA糖基酶，它切除烷基化的碱基以创建基本位点并启动碱基切除修复(BER)途径。DNA烷基化修复是基因组稳定性的关键，也是癌症化疗的主要耐药因素，因此其他研究较少但具有生物学意义的关键烷基化修复途径值得研究。因此，这一建议侧重于重要的非糖基酶途径，通过直接逆转(目标1)或通过非经典地将损伤引导到主要DNA切除修复途径之一(目标2-4)的途径“串扰”蛋白质来消除烷基化损伤，以避免有毒DNA物种的释放。到目前为止，我们的工作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同系物3)的结构化学，并支持了目标1中提出的进一步的特征。此外，我们还发现了三个系统来表征串扰，这是烷基化修复途径交叉的重要细胞策略，促进受损DNA非经典地进入切除修复途径。因此，我们将进一步鉴定三种特定的烷基化碱基损伤反应蛋白，它们促进非经典的进入DNA切除修复的三条原型途径中的每一条：Aim 2)ATL(烷基转移酶样蛋白)，它是转移酶不活跃的，但在基因上与核苷酸切除修复(NER)有关，它切除扭曲DNA的巨大病变，Aim 3)AGTdoV(O6-烷基鸟嘌呤-DNA-烷基转移酶-核酸内切酶)，它共价连接AGT和Endo V DNA主干切除酶，形成作为BER底物的断裂，目的4)糖基酶失活的MAG2(甲基腺嘌呤糖基酶同系物2)，它在基因和结构上与错配修复(MMR)相连，错配修复通常会切除错配区域。我们建议将Tainer实验室的大分子X射线结晶学(MX)和溶液中小角X射线散射(SAXS)对蛋白质和复合体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相结合。这项拟议的工作将对核心烷基化修复起始蛋白及其在体内的功能进行表征，以阐明非糖基酶烷基化损伤修复关键方面的结构-功能机制。总体而言，这些结果将提供对与遗传完整性、化疗耐药性相关的烷基化损伤反应的统一理解，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烷基化修复蛋白及其抑制剂，以及与新的治疗策略和癌症化疗相关的步骤。与公共卫生相关DNA烷基化是导致癌症易感性的基因组不稳定的来源之一，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招募非经典修复机器来纠正损伤来直接消除；然而，这些途径所介导的结构化学和“串扰”机制都不是完全清楚的。我们建议对烷基化损伤修复的这两个关键方面的结构细胞生物学进行表征，这两个方面与改进的癌症化疗和环境制剂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.