Molecular Architecture of Oxidative Stress Induced Double Strand Break Repair

氧化应激诱导双链断裂修复的分子结构

• 批准号: 10755883
• 负责人: JOONAS JAMSEN
• 金额: $ 24.9万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-08 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10755883
• 关键词: 8-hydroxyguanosine; 8-oxo-dGTP; Active Sites; Address; Adenine; Aging; Air; Air Pollution; Architecture; Base Excision Repairs; Base Pairing; Behavior; Biological Assay; Bypass; Catalysis; Cells; Chemicals; Complex; Cryoelectron Microscopy; Crystallography; DNA; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA biosynthesis; DNA lesion; DNA polymerase mu; DNA-Directed DNA Polymerase; Deoxyguanosine; Development; Disease; Disease Progression; Double Strand Break Repair; Environment; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Etiology; Exposure to; Food; Future; Genome; Genome Stability; Genomic Instability; Goals; Guanosine Triphosphate; Hydrogen Bonding; Kinetics; Knowledge; Laboratories; Lead; Lesion; Link; Maintenance; Malignant Neoplasms; Mediating; Methods; Mission; Molecular; Molecular Analysis; Multiprotein Complexes; Mutagenesis; Mutation; National Institute of Environmental Health Sciences; Neurodegenerative Disorders; Nitrogen; Nonhomologous DNA End Joining; Nucleotides; Outcome; Oxidative Stress; Oxidative Stress Induction; Oxygen; Phase; Polymerase; Population; Predisposition; Productivity; Proteins; Radiation; Reaction; Reactive Oxygen Species; Repair Complex; Research; Role; Structure; Training; Visualization; Water; Work; artemis; base; cancer risk; career; effective therapy; environmental agent; environmental mutagens; enzyme substrate; genome integrity; human disease; insight; mutant; new technology; normal aging; oxidative DNA damage; oxidative damage; prevent; protein complex; repaired; targeted cancer therapy; therapy development; transversion mutation

Abstract Excess reactive oxygen and nitrogen species (RONS) from radiation, polluted air, as well as chemicals in food and water induce oxidative stress and cause direct damage to nucleotide bases. A prominent form of oxidized damage in the genome and free nucleotide pools is 8-oxoG or 8-oxo-dGTP, respectively. 8-oxoG can hydrogen bond with adenine, resulting in mutations upon DNA replication. 8-oxoG also produces double strand breaks (DSBs) that undergo error-prone repair through non-homologous end-joining (NHEJ). This results in mutagenesis and genomic instability. DNA polymerases mediate repair of oxidative DNA damage. A major obstacle to understanding the etiology and progression of diseases caused by oxidative stress is that the DNA polymerase mechanism employed in the repair of oxidative lesions and effects of accessory factors in the DNA repair complex remain poorly understood. Solving this problem will enable a more complete understanding of the role of DNA polymerases in cancer, aging and disease. The knowledge gained will enable development of therapies to target cancer, neurodegenerative disorders and aging. I will uncover the impact of oxidative stress-induced DNA repair on genome integrity by determining how active site contacts and dynamics in polymerases influence repair outcomes. I will also relate my observations to accessory repair factors and larger repair complexes. To accomplish this task, I will use pH jump crystallography to determine snapshots that will reveal the active site contacts and dynamics employed by NHEJ polymerases λ and μ to insert and extend from an 8-oxoG lesion (Aim 1, K99 phase). The functional significance of these contacts will be verified using kinetic assays and mutant enzymes. I hypothesize that these contacts regulate the unique behavior of these polymerases in promoting productive and accurate synthesis and provide insight into the role of 8-oxoG in mutagenesis. I will then employ a combination of transient kinetics and time- lapse crystallography to determine the atomic level contacts and dynamics employed by polymerases λ and μ to perform translesion synthesis past and proofreading of the 8-oxoG lesion (Aim 2, R00 phase). The structural intermediates determined in this aim will allow understanding of the structural requirements for mutation prone bypass of 8-oxoG. Building on the results of the latter two aims and training during the K99 period, I will determine the role of substrate channeling among accessory factors during oxidative DNA damage induced NHEJ (Aim 3, R00 phase). Cryo-EM studies will enable determination of how the dynamic and heterogenous populations of NHEJ repair complexes impact oxidative DNA damage repair. I will also determine the structural basis for 8- oxoG processing by Artemis during oxidative NHEJ repair. Completion of this aim will require training in Cryo- EM that will be provided by Dr. Mario Borgnia. The knowledge gained as a result of the work on how polymerases impact repair of oxidative damage and how this mediates productive repair will provide a significant advance in the understanding of how environmental agent induced DNA damage repair. I will gain expertise in state-of-the-art methods such as crystallography, transient kinetics, cryo-EM, DNA replication and DNA repair that will help me achieve my career goals and establish an independent laboratory.

抽象的 辐射、污染空气以及食品中的化学物质导致过量的活性氧和氮 (RONS) 水会引起氧化应激，并对核苷酸碱基造成直接损害，这是一种重要的氧化形式。 基因组和游离核苷酸库中的损伤分别是8-oxoG或8-oxo-dGTP，8-oxoG可以氢。 与腺嘌呤结合，导致 DNA 复制时发生突变，也会产生双链断裂。 （DSB）通过非同源末端连接（NHEJ）进行容易出错的修复。 突变和基因组不稳定性。 DNA聚合酶介导氧化DNA损伤的修复是理解DNA聚合酶的主要障碍。 氧化应激引起的疾病的病因和进展在于DNA聚合酶机制 DNA修复复合物中氧化损伤修复中的作用和辅助因子的作用仍然存在 解决这个问题将使人们更全面地了解 DNA 的作用。 所获得的知识将有助于开发针对癌症、衰老和疾病的聚合酶。 癌症、神经退行性疾病和衰老。 我将通过确定氧化应激诱导的 DNA 修复对基因组完整性的影响来揭示氧化应激诱导的 DNA 修复对基因组完整性的影响。 聚合酶中的活性位点接触和动态会影响修复结果。 为了完成这项任务，我将使用 pH 跳跃。 晶体学以确定快照，从而揭示 NHEJ 使用的活性位点接触和动力学 聚合酶 λ 和 μ 插入 8-oxoG 损伤并从其延伸（目标 1，K99 相）。 这些接触将使用动力学测定和突变酶进行验证。 调节这些聚合酶在促进高效和准确合成方面的独特行为，并提供 然后，我将结合瞬态动力学和时间动力学来深入了解 8-oxoG 在诱变中的作用。 延时晶体学测定聚合酶 λ 和 μ 所采用的原子级接触和动力学 对 8-oxoG 病变进行跨损伤合成和校对（目标 2，R00 阶段）。 为此目的确定的中间体将有助于了解易突变的结构要求 根据后两个目标和 K99 期间训练的结果，我将确定 8-oxoG 的绕过。 辅助因子之间的底物通道在 DNA 氧化损伤诱导的 NHEJ 过程中的作用（目标 3， R00 相）。冷冻电镜研究将能够确定动态和异质群体的情况。 NHEJ 修复复合物影响氧化 DNA 损伤修复。我还将确定 8- 的结构基础。 Artemis 在氧化 NHEJ 修复过程中进行 oxoG 处理 完成此目标需要接受冷冻培训。 EM 将由 Mario Borgnia 博士提供。 通过研究聚合酶如何影响氧化损伤修复而获得的知识 以及这如何调解生产性修复将在理解如何 我将获得最先进方法的专业知识，例如 晶体学、瞬态动力学、冷冻电镜、DNA 复制和 DNA 修复将帮助我实现我的职业生涯 目标并建立一个独立的实验室。