Molecular Architecture of Oxidative Stress Induced Double Strand Break Repair

摘要 辐射、污染的空气以及食物中的化学物质所产生的过量的活性氧和氮(RON) 水会引起氧化应激，并对核苷酸碱基造成直接损害。一种突出的氧化形式 基因组和游离核苷酸池中的损伤分别是8-oxoG或8-oxo-dGTP。8-oxoG罐头氢气 与腺嘌呤结合，导致DNA复制时发生突变。8-oxoG还会产生双链断裂 (DSB)，通过非同源末端连接(NHEJ)进行错误修复。这将导致 突变和基因组的不稳定性。 DNA聚合酶介导DNA氧化损伤的修复。理解这个问题的一个主要障碍 氧化应激引起的疾病的病因和进展是DNA聚合酶机制 用于修复氧化损伤和辅助因子在DNA修复复合体中的作用 人们对此知之甚少。解决这个问题将使我们能够更全面地了解DNA的作用 聚合酶与癌症、衰老和疾病。所获得的知识将使治疗方法的开发能够达到目标 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，方法是确定 聚合酶中的活性部位接触和动态影响修复结果。我还将讲述我的观察结果 到附属修复因子和较大的修复复合体。为了完成这项任务，我将使用pH跳跃 结晶学，以确定将揭示NHEJ采用的活性部位接触和动力学的快照 聚合酶λ和μ从8-oxoG病变插入和延伸(目标1，K99相)。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高产和准确合成方面的独特行为，并提供 洞察8-oxoG在诱变中的作用。然后我将使用瞬变动力学和时间的组合- 测定聚合酶λ和μ使用的原子级接触和动力学的跃迁结晶学 对8-oxoG损伤(目标2，R00期)进行跨病变合成、过去和校对。结构性的 在这一目标中确定的中间体将允许理解易于突变的结构要求 8-氧葡萄糖搭桥术。根据后两个目标的结果和K99期间的训练，我将决定 底物通道在氧化DNA损伤诱导NHEJ过程中的作用(目标3， R00阶段)。冷冻-EM研究将能够确定动态和异质的种群如何 NHEJ修复复合体影响DNA氧化损伤修复。我还将确定8-8的结构基础- Artemis在NHEJ氧化修复过程中的oxoG处理。完成这一目标将需要培训低温- EM将由Mario Borgnia博士提供。 聚合酶如何影响氧化损伤修复的工作所获得的知识 以及这如何促进生产性修复，将大大促进对如何 环境因子诱导DNA损伤修复。我将获得最先进的方法的专业知识，例如 结晶学、瞬变动力学、冷冻-EM、DNA复制和DNA修复，这将帮助我实现我的职业生涯 目标和建立一个独立的实验室。

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.