Integrative Structural Biology in DNA Replication and Damage Response

DNA 复制和损伤反应中的综合结构生物学

• 批准号: 10330665
• 负责人: WALTER J. CHAZIN
• 金额: $ 43.59万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330665
• 关键词: Address; Automobile Driving; Binding Proteins; Biochemical; Biophysics; Cells; Collaborations; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Primase; DNA Replication Damage; DNA biosynthesis; DNA polymerase alpha-primase; DNA replication fork; Daughter; Defect; Development; Disease; Exposure to; Genome; Genomic Instability; Goals; Hand; Knowledge; Lead; Length; Maintenance; Malignant Neoplasms; Modeling; Motor; Multiprotein Complexes; Mutation; Oxidation-Reduction; Pathway interactions; Patients; Play; Polymerase; Process; Property; Proteins; RNA chemical synthesis; Role; Signal Transduction; Structure; Sunlight; Testing; Toxic Environmental Substances; biophysical properties; delta opioid receptor; insight; mutant; recruit; response; structural biology; targeted treatment; three dimensional structure

PROJECT SUMMARY Faithful replication of DNA and response to encounters with aberrant DNA are essential to cell propagation and survival. Our long-term goal is to understand the action of multi-protein DNA replication and damage response machinery at eukaryotic replication forks. Our strategy is to elucidate the structural mechanisms using an integrative structural biology approach, coupled to biochemical/biophysical characterization and collaborations to define functional implications. This proposal focuses on critical unsolved questions about the initiation of daughter strand synthesis in replication, and the stalling and remodeling of replication forks upon encountering aberrant DNA. In DNA replication, the processive polymerases δ and ε require a short primer strand on the template to function, which is generated by DNA polymerase a-primase (pol-prim). Although 3D structures have been determined for all components of pol-prim and even the intact heterotetramer, these have provided only limited mechanistic insights because structures of the full-length protein with relevant substrates and essential co-factors are lacking. To address this critical gap in knowledge, we propose to determine the relevant structures using Cryo-EM. We also propose to continue working on characterizing the structure, biochemical properties and functional roles of 4Fe-4S clusters in pol-prim. We will test and refine our hypotheses about the role of: (i) the primase 4Fe-4S cluster redox in modulating DNA binding activity; (ii) the role of the cluster in pol α in driving the transition from RNA synthesis by primase to DNA synthesis by pol α. Together, these studies will solve the fundamental questions about how pol-prim counts the length of the primer at each step and how the substrate hand-offs occur from primase to pol α and then from pol α to pols δ or ε. Our second project addresses two critical gaps in knowledge about replication fork encounters with aberrant DNA. RPA and Rad51 are two highly abundant ssDNA binding proteins that have critical roles in the stalling, reversal and stabilization of stalled forks. RPA-coated ssDNA is the key initiating signal for multiple damage response pathways and plays several additional roles, including recruiting and directing the fork reversal activity of the ATP motor protein SMARCAL1. We propose to elucidate the mechanisms that drive this important aspect of fork remodeling by determining the structure of the RPA and SMARCAL1 on a model fork substrate complex using Cyro-EM. Rad51 plays an essential role in the stabilization of stalled replication forks. Collaborative studies with David Cortez led to the discovery and characterization of RADX, a new DNA damage response protein involved in regulating the activity of Rad51 at stalled forks. We recently discovered RADX also interacts physically with RPA, suggesting there is a RPA-RADX-Rad51 network operating at stalled forks. We propose combined structural, biophysical and functional analyses of RADX and its interactions with DNA, Rad51 and RPA to clarify the roles of RADX at stalled replication forks. Together, our two projects will greatly enhance understanding of how DNA is processed at eukaryotic replication forks and genomes are maintained and propagated.

项目概要 DNA 的忠实复制和对异常 DNA 的反应对于细胞增殖和发育至关重要。 生存。我们的长期目标是了解多蛋白 DNA 复制和损伤反应的作用 真核复制叉的机器。我们的策略是利用 综合结构生物学方法，结合生化/生物物理表征和合作 定义功能含义。该提案重点关注有关启动的关键未解决问题 复制中的子链合成，以及遇到复制叉时的停滞和重塑 异常DNA。在 DNA 复制中，持续聚合酶 δ 和 ε 需要短引物链 功能模板，由 DNA 聚合酶 a-primase (pol-prim) 生成。尽管 3D 结构具有 已确定 pol-prim 的所有成分，甚至完整的异四聚体，这些仅提供了 机制见解有限，因为全长蛋白质的结构具有相关底物和必需的 缺乏辅助因素。为了解决这一知识上的关键差距，我们建议确定相关结构 使用冷冻电镜。我们还建议继续致力于表征结构、生化特性 以及 pol-prim 中 4Fe-4S 簇的功能作用。我们将测试并完善我们关于以下角色的假设：(i) 引物酶 4Fe-4S 簇氧化还原调节 DNA 结合活性； (ii) pol α 中的簇在驱动中的作用 从引物酶合成RNA到pol α合成DNA的转变。这些研究共同将解决 关于 pol-prim 如何在每一步计算引物长度以及底物如何计算的基本问题 交接发生从引物酶到 pol α，然后从 pol α 到 pol δ 或 ε。我们的第二个项目解决了两个问题 关于复制叉与异常 DNA 相遇的知识存在重大差距。 RPA 和 Rad51 是两个高度 丰富的 ssDNA 结合蛋白，在失速叉的失速、逆转和稳定中发挥关键作用。 RPA 包被的 ssDNA 是多种损伤反应途径的关键启动信号，并发挥多种作用 其他作用，包括招募和指导 ATP 运动蛋白 SMARCAL1 的叉逆转活动。 我们建议通过确定驱动前叉重塑这一重要方面的机制来阐明 使用 Cyro-EM 显示叉基底复合物模型上的 RPA 和 SMARCAL1 结构。 Rad51 扮演 在稳定停滞的复制叉中发挥着重要作用。与 David Cortez 的合作研究导致 RADX 的发现和表征，这是一种参与调节活性的新型 DNA 损伤反应蛋白 Rad51 在停滞的货叉处。我们最近发现 RADX 也与 RPA 进行物理交互，这表明 在停滞的分叉上运行的 RPA-RADX-Rad51 网络。我们建议结合结构、生物物理和 RADX 的功能分析及其与 DNA、Rad51 和 RPA 的相互作用，以阐明 RADX 在停滞时的作用 复制叉。我们的两个项目将共同极大地加深人们对 DNA 是如何加工的理解。 真核复制叉和基因组得以维持和繁殖。