Molecular Architecture of Oxidative Stress Induced Double Strand Break Repair

摘要 过量的活性氧和氮物质（RONS）来自辐射，污染的空气以及食品中的化学物质 和水诱导氧化应激并导致核苷酸碱基的直接损伤。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。8-oxoG也产生双链断裂 通过非同源末端连接（NHEJ）进行易错修复的DSB。这导致 诱变和基因组不稳定性。 DNA聚合酶介导氧化性DNA损伤的修复。理解的主要障碍 由氧化应激引起的疾病的病因学和进展是DNA聚合酶机制 用于修复氧化损伤和辅助因子在DNA修复复合物中的作用 不太了解。解决这个问题将使人们能够更全面地了解DNA的作用 聚合酶在癌症、衰老和疾病中的作用。所获得的知识将有助于开发针对 癌症、神经退行性疾病和衰老。 我将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力学影响修复结果。我也会把我的观察 辅助修复因子和更大的修复复合体。为了完成这项任务，我将使用pH值跳跃 晶体学，以确定快照，将揭示活性位点接触和动力学所采用的NHEJ 在一些实施方案中，聚合酶λ和μ能够从8-oxoG损伤插入和延伸（Aim 1，K99期）。的功能意义 将使用动力学分析和突变酶来验证这些接触。我假设这些联系人 调节这些聚合酶在促进生产性和精确合成中的独特行为， 深入了解8-oxoG在突变中的作用。我将结合瞬态动力学和时间- 用于确定聚合酶λ和μ所采用的原子水平接触和动力学的晶体学 进行8-oxoG损伤的跨损伤合成和校对（Aim 2，R 00阶段）。结构性 在这个目标中确定的中间体将允许理解突变倾向的结构要求。 旁路8-oxoG。根据后两个目标的结果和K99期间的培训，我将确定 在NHEJ诱导的氧化性DNA损伤过程中辅助因子之间的底物通道作用（Aim 3， R 00相）。冷冻EM研究将能够确定如何动态和异质群体的 NHEJ修复复合物影响氧化DNA损伤修复。我还将确定8- Artemis在氧化性NHEJ修复期间进行oxoG加工。要实现这一目标，就需要进行低温培训。 将由Mario Borgnia博士提供EM。 从聚合酶如何影响氧化损伤修复的工作中获得的知识 以及这是如何介导生产性修复的，将为理解如何 环境因子诱导的DNA损伤修复。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，这将帮助我实现我的职业生涯 并建立独立实验室。

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.