Structural Biochemistry of DNA Dealkylation

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， 典型地由损伤特异性DNA糖基化酶修复，该酶切除烷基化碱基， 碱基切除修复（BER）途径。DNA烷基化修复对于 基因组稳定性，而且是癌症化疗的主要耐药因素，因此其他 较少研究但生物学上关键的烷基化修复途径值得表征。因此，这一建议 侧重于重要的非糖基化途径，其中烷基化损伤是通过直接 逆转（目标1），或通过途径“串扰”蛋白，非经典引导损伤进入其中之一， 主要的DNA切除修复途径（目的2-4），以避免释放有毒的DNA物质。我们努力 数据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），以及 支持他们在目标1中提出的进一步表征。我们还发现了三个系统， 表征串扰，一种重要的烷基化修复途径交叉的细胞策略， 促进受损DNA非经典进入切除修复途径。因此我们将 此外，还表征了三种特异性烷基化碱基损伤反应蛋白， DNA切除修复的三种原型途径中的每一种的经典入口：Aim 2）ATL（烷基- 转移酶样），其是转移酶失活的，但在遗传上与核苷酸切除修复相关 (NER)Aim 3）AGTendoV（O6-alkylguanine-DNA- 烷基转移酶-内切核酸酶V），其将AGT与Endo V DNA骨架共价连接 切除酶以形成作为BER底物的断裂，和Aim 4）糖基化酶失活的Mag 2 （甲基腺嘌呤糖基化酶同系物2），在遗传和结构上与错配修复相关 (MMR)切除不匹配的区域。我们建议将定量生物物理 通过大分子X射线晶体学（MX）和小分子X射线晶体学（X-ray crystallography）表征蛋白质和复合物 在Tainer实验室进行的溶液中的角X射线散射（SAXS）， 佩格实验室的体内生化和突变结果拟议的工作将以 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的关键方面的机制。总的来说，这些结果将 提供与遗传完整性相关的烷基化损伤反应的统一理解， 化疗抗性，以及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，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性的来源，并且也是导致癌症易感性的主要因素。 癌症化疗的结果。烷基化损伤可以通过逆转碱损伤或 通过招募非经典修复机制来纠正病变;然而，结构化学 也不完全理解由这些途径介导的“串扰”的机制。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， 与改进癌症化疗和环境因子风险评估有关。

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.