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: 10809376
• 负责人: WALTER J. CHAZIN
• 金额: $ 1.43万
• 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/10809376
• 关键词: Address; Automobile Driving; Binding Proteins; Biochemical; Biophysics; Cells; Collaborations; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Primase; DNA biosynthesis; DNA polymerase alpha-primase; DNA replication fork; Defect; Development; Disease; Exposure to; Genome; Genomic Instability; Goals; 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; Single-Stranded DNA; Structure; Sunlight; Testing; Toxic Environmental Substances; biophysical properties; cofactor; daughter strand; 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 α-primase (pol-prim). Although 3D structures have been determined for all components of pol-prim and the intact heterotetramer, these have provided only limited mechanistic insights because structures of 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) primase 4Fe-4S cluster redox in modulating DNA binding activity; (ii) the role of the cluster in polα 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 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 initiating signal for 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 Cryo-EM. Rad51 plays an essential role in the stabilization of stalled replication forks. Collaborative studies led to the discovery and characterization of RADX, a new DNA damage response protein involved in regulating the activity of Rad51 at stalled forks. 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至关重要 我们的长期目标是了解多蛋白 DNA 复制和损伤的作用。 我们的策略是阐明真核复制叉的结构机制。 使用综合结构生物学方法，结合生化/生物物理表征和 该提案重点关注有关未解决的关键问题。 复制中子链合成的起始，以及复制叉的停滞和重塑 遇到异常 DNA 在 DNA 复制中，持续聚合酶 δ 和 ε 需要短引物。 模板上的链发挥作用，它是由 DNA 聚合酶 α-primase (pol-prim) 产生的，尽管是 3D。 已确定 pol-prim 和完整异四聚体的所有成分的结构，这些已 由于全长蛋白质与相关底物的结构，仅提供了有限的机制见解 为了解决这一知识上的关键差距，我们建议确定 我们还建议继续致力于表征结构， 我们将测试和完善 pol-prim 中 4Fe-4S 簇的生化特性和功能作用。 关于以下作用的假设：(i) 引物酶 4Fe-4S 簇氧化还原在调节 DNA 结合活性中的作用； polα 中的簇驱动从引物酶合成 RNA 到 pol α 合成 DNA 的转变。 这些研究将共同​​解决有关 pol-prim 如何计算长度的基本问题。 每个步骤的引物以及底物如何从引物酶转移到 pol α，然后从 pol α 到 pols δ 或 ε 我们的第二个项目解决了有关复制叉遭遇的两个关键知识空白。 RPA 和 Rad51 是两种高度丰富的 ssDNA 结合蛋白，在 RPA 包被的 ssDNA 的停滞、逆转和稳定是损伤的起始信号。 反应途径并发挥一些额外的作用，包括招募和指导分叉逆转 我们建议阐明 ATP 运动蛋白 SMARCAL1 的驱动机制。 通过确定模型前叉上 RPA 和 SMARCAL1 的结构来进行前叉重塑的一个重要方面 使用 Cryo-EM 的底物复合物在稳定复制停滞中发挥着重要作用。 合作研究导致了 RADX（一种新的 DNA 损伤反应）的发现和表征。 参与调节停滞叉处 Rad51 活性的蛋白质也与 RPA 发生物理相互作用。 表明有一个 RPA-RADX-Rad51 网络在停滞的分叉上运行。我们建议组合结构， 对 RADX 及其与 DNA、Rad51 和 RPA 的相互作用进行生物物理和功能分析，以阐明 我们的两个项目将共同极大地增强对 RADX 在停滞复制叉中的作用的理解。 DNA 在真核复制叉上如何加工以及基因组如何维持和传播。