Structural Biochemistry of DNA Dealkylation

DNA 脱烷基化的结构生物化学

• 批准号: 7496688
• 负责人: John A. Tainer
• 金额: $ 9.27万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7496688
• 关键词: Address; Affinity; Alkylation; Animal Model; Bacteria; Base Excision Repairs; Binding; Biochemical; Biochemistry; Catalysis; Chemicals; Chemotherapy-Oncologic Procedure; Complement; Complex; Computing Methodologies; Crystallography; DNA; DNA Alkylation; DNA Binding; DNA Damage; DNA Modification Process; DNA Repair; DNA glycosylase; DNA lesion; DNA-Binding Proteins; Dealkylation; Detection; Development; Dioxygenases; Excision; Figs - dietary; Genetic; Genetic Screening; Genome Stability; Genomic Instability; Human; In Vitro; Institution; Lesion; Light; Macromolecular Complexes; Mediating; Mismatch Repair; Molecular; Pathway interactions; Proteins; Regulation; Research; Research Personnel; Resistance; Role; Scanning; Site; Solutions; Source; Specificity; Staging; Structural Biochemistry; Structural Chemistry; Sum; System; Techniques; Testing; Therapeutic; Translating; Work; Yeasts; alkyltransferase; base; cancer therapy; chemotherapy; comparative; concept; cytotoxic; in vivo; inhibitor/antagonist; microbial; novel; novel therapeutics; programs; protein structure; repaired; research study; resistance factors; response

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.

烷基化DNA碱基损伤是细胞毒性和致突变的，除非修复，并且是大多数DNA的原型 涉及DNA碱基的化学修饰的损伤。因此，DNA烷基化修复对于 基因组稳定性，并且是癌症化疗的主要抗性因素。DNA-糖基化酶 通过碱基切除修复（BER）去除烷基化碱基的方法相对较好地表征。然而两 烷基化损伤修复的关键方面仍然知之甚少：1） 烷基化损伤反转和2）连接烷基化基极损伤响应到其它修复的串扰 途径。总的来说，该项目的两个具体目标将解决在 分子水平的主要烷基化损伤逆转蛋白在人类，和关键的串扰连接 对其他修复途径的烷基化损伤。目标尽可能以人类系统为中心， 在需要的地方使用模式生物，以揭示保守DNA的核心结构生物化学 修理机器。我们建议对这些知之甚少的反转和串扰修复进行表征 通过整合化学，突变和生物化学方法与两种互补机制， 大分子X射线晶体学（MX）和小角X射线散射（SAXS）的结构技术， 溶液因此，这项工作将适当地利用和整合研究成果，优势， 研究人员及其机构的计划，以促进，发展，并测试统一的理解， 烷基化损伤反应蛋白和新的抑制剂。蛋白质结构的定量表征 和复合物的MX和SAXS沿着与生物物理方法和计算分析在泰纳实验室 将与Pegg实验室的详细体外和体内生化和突变结果相协调。在 我们的分析，我们将解决三个基本假设：1）损害特异性来自总和 - 多个子步骤，其促进底物识别，这可以通过实验来剖析， 表征了2)初始阶段为多步修复途径提供承诺。这些阶段包括 构象变化，形成稳定的DNA产物复合物，因此顺序编排 修复步骤部分由结合亲和力和界面交换控制。3)蛋白质可以控制串扰 并通过DNA雕刻与其他修复途径连接，以创建招募平台， 通过启动另一种不同修复途径的蛋白质促进DNA结合。这些总结的想法 特异性步骤、用于连续裂解的蛋白质-DNA产物复合物和蛋白质指导的DNA雕刻 促进通路连接的作用。因此，这些概念指导我们 目前的努力，破译动态相互作用的烷基化基地逆转和修复蛋白质。总的来说，这些 结果将提供一个统一的理解烷基化损伤反应相关的表征他们的作用 在遗传完整性和对化疗药物的抗性方面，以及促进癌症治疗的进展。