Structural Biochemistry of DNA Dealkylation

烷基化的DNA碱基损伤是细胞毒性和突变性的，除非修复，而且是大多数DNA的原型 涉及DNA碱基化学修饰的损伤。因此，DNA烷基化修复对于 基因组的稳定性，而且是癌症化疗的主要耐药因素。DNA糖基酶 通过碱基切除修复(BER)去除烷基化碱基的研究相对较好。然而，有两个 烷基化损伤修复的关键方面仍然知之甚少：1)结构化学 烷基化损伤逆转和2)串扰连接烷基化碱基损伤对其他修复的反应 小路。总体而言，该项目的两个具体目标将解决在 分子水平上，人类主要的烷基化损伤逆转蛋白，以及关键的串扰连接 烷基化对其他修复途径的破坏。目标是尽可能以人类系统为中心，并 在需要的地方使用模型生物来揭示保存完好的DNA的核心结构生物化学 修理机器。我们建议将这些鲜为人知的反转和串扰修复描述为 通过将化学、突变和生化方法与两种互补的方法相结合来实现机制 大分子X射线结晶学(MX)和小角X射线散射(SAXS)的结构技术 解决方案。因此，这项工作将适当地利用和整合研究成果、优势、 以及调查人员及其机构的计划，以促进、发展和检验对 烷基化损伤反应蛋白和新型抑制剂。蛋白质结构的定量表征 以及Tainer实验室的生物物理方法和计算分析 将与来自佩格实验室的详细的体外和体内生化和突变结果相协调。在……里面 在我们的分析中，我们将解决三个基本假设：1)损害特异性来自于 促进底物识别的多个子步骤，可以通过实验解剖和 特色化的。2)初始阶段提供了对多步骤修复途径的承诺。这些阶段包括 构象变化形成稳定的DNA产物复合体用于移交，因此顺序编排 修复步骤在一定程度上受绑定亲和力和接口交换的控制。3)蛋白质可以控制串扰 以及通过DNA雕刻与其他修复途径的连接，以创建招聘平台 通过启动另一条不同的修复途径的蛋白质促进DNA结合。这些观点总结了 特定步骤、用于顺序切换的蛋白质-DNA产物复合体和蛋白质指导的DNA雕刻 从我们的结果已经阐明了促进通路连接的作用。因此，这些概念指导我们 目前正在努力破译烷化碱基逆转和修复蛋白的动态相互作用。总的来说，这些 结果将提供对与表征其作用相关的烷基化损伤反应的统一理解 在遗传完整性和对化疗药物的抵抗力方面，以及在促进癌症治疗方面的进展。

Alkylated DNA base damage is cytotoxic and mutagenic unless repaired, and is prototypic of most DNA damage involving the chemical modification of DNA bases. DNA alkylation repair is therefore critical for genome stability and is furthermore a major resistance factor for cancer chemotherapies. DNA-glycosylases that remove alkylated bases by base-excision repair (BER) are relatively well characterized. However, two critical aspects of alkylation damage repair are still poorly understood: 1) the structural chemistries for alkylation damage reversal and 2) the crosstalk connecting alkylated base damage responses to other repair pathways. Overall, the two Specific Aims of this Project will address the challenge of characterizing at the molecular level the major alkylation damage reversal proteins in humans, and the critical crosstalk connecting alkylation damage to other repair pathways. The aims center on the human systems where possible and employ model organisms where needed, to reveal the core structural biochemistry of the well-conserved DNA repair machinery. We propose to characterize these poorly understood reversal and crosstalk repair mechanisms by integrating chemical, mutational, and biochemical approaches with two complementary structural techniques of macromolecular x-ray crystallography (MX) and small angle x-ray scattering (SAXS) in solution. Thus, this work will appropriately leverage and integrate the research accomplishments, strengths, and programs of the investigators and their institutions to promote, develop, and test a unified understanding of alkylation damage response proteins and novel inhibitors. Quantitative characterization of protein structures and complexes by MX and SAXS along with biophysical methods and computational analyses in the Tainer lab will be coordinated with detailed in vitro and in vivo biochemical and mutational results from the Pegg lab. In our analyses, we will address three fundamental hypotheses: 1) Damage specificity comes from the sum of multiple sub-steps that promote substrate recognition, which can be experimentally dissected and characterized. 2) Initial stages provide commitment to the multi-step repair pathways. These stages involve conformational changes forming stable DNA product complexes for handoffs, so sequential orchestration of repair steps is in part governed by binding affinity and interface exchanges. 3) Proteins can control crosstalk and connections with other repair pathways through DNA sculpting, to create recruitment platforms that promote DNA binding by proteins that initiate another distinct repair pathway. These ideas of summed specificity steps, protein-DNA product complexes for sequential handoffs, and protein-directed DNA sculpting to promote pathway connections have been elucidated from our results. These concepts therefore guide our current efforts to decipher the dynamic interplay of alkylated base reversal and repair proteins. Overall, these results will provide a unified understanding of alkylation damage responses relevant to characterizing their role in genetic integrity and resistance to chemotherapeutics, and to promoting advances in cancer therapies.