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: 10796477
• 负责人: WALTER J. CHAZIN
• 金额: $ 7.27万
• 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/10796477
• 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 replication fork; DNA-Directed DNA Polymerase; 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 -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.

项目摘要 DNA的忠实复制和对遇到异常DNA的反应对于细胞繁殖和增殖是必不可少的。 生存我们的长期目标是了解多蛋白质DNA复制和损伤反应的作用 真核生物复制叉上的机器。我们的策略是使用一个 综合结构生物学方法，结合生物化学/生物物理表征和合作 定义功能含义。这项建议的重点是关于启动 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，进行性聚合酶δ和ε需要短引物链上的引物。 模板的功能，这是由DNA聚合酶β-引物酶（pol-primase）产生的。虽然3D结构具有 已确定的所有组成部分的pol-benzene，甚至完整的异源四聚体，这些只提供了 有限的机制的见解，因为全长蛋白质的结构与相关的底物和必要的 缺乏辅助因素。为了解决这一关键的知识差距，我们建议确定相关的结构， 使用冷冻电镜我们还建议继续研究其结构、生化特性和生物活性。 以及4Fe-4S团簇在极化过程中的功能作用。我们将测试和完善我们的假设的作用：（一） 引发酶4Fe-4S簇氧化还原在调节DNA结合活性中的作用;（ii）pol α簇在驱动DNA结合活性中的作用 从引物酶合成RNA到聚合酶α合成DNA的转变。这些研究将共同解决 基本问题是pol-technology如何在每一步计算引物的长度，以及底物如何 切换发生在从primase到pol α，然后从pol α到pol δ或ε。我们的第二个项目涉及两个 关于复制叉遇到异常DNA的知识存在重大空白。RPA和Rad 51是两个高度 丰富的ssDNA结合蛋白，在停滞，逆转和稳定停滞叉中起关键作用。 RPA包被的ssDNA是多个损伤反应途径的关键起始信号，并发挥多个作用。 其他作用，包括招募和指导ATP马达蛋白SMARCAL 1的叉逆转活性。 我们建议通过确定这些因素来阐明推动叉子重塑这一重要方面的机制。 使用Cyro-EM的RPA和SMARCAL 1在模型叉基底复合物上的结构。Rad 51扮演一个 在稳定停滞的复制叉方面发挥着至关重要的作用。与大卫科尔特斯的合作研究导致了 一种新的DNA损伤反应蛋白RADX的发现和鉴定 Rad 51在失速的岔路口。我们最近发现RADX也与RPA发生物理相互作用，这表明 RPA-RADX-Rad 51网络在失速分叉处运行。我们建议结合结构，生物物理和 RADX的功能分析及其与DNA，Rad 51和RPA的相互作用，以阐明RADX在失速时的作用 复制分叉。我们的两个项目将大大增强对DNA如何加工的理解 真核生物的复制叉和基因组得以维持和繁殖。