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的忠实复制和对异常DNA相遇的反应对于细胞传播至关重要 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 真核复制叉的机械。我们的策略是使用 综合结构生物学方法，结合生化/生物物理特征和协作 定义功能含义。该提案着重于有关该计划的关键尚未解决的问题 遇到复制的女儿链综合，复制叉的失速和重塑 异常DNA。在DNA复制中，过程聚合酶δ和ε需要在 功能的模板，该模板是由DNA聚合酶-原则酶（POL-PRIM）生成的。尽管有3D结构 已确定POL-PRIM甚至完整的异光学器的所有组件，它们仅提供 机械见解有限，因为全长蛋白质的结构具有相关的底物和必不可少的 缺乏联合因素。为了解决知识的关键差距，我们建议确定相关结构 使用Cryo-Em。我们还建议继续努力表征结构，生化特性 4FE-4S簇在POL-PRIM中的功能作用。我们将测试和完善有关：（i）的作用的假设 Primase 4FE-4S簇氧化还原在调节DNA结合活性中； （ii）簇在polα中的作用在驾驶中 从原始酶从RNA合成到polα合成的DNA合成的过渡。这些研究将共同​​解决 关于POL-POLIM如何计算每个步骤底漆的长度以及底物的基本问题 从原始酶到polα，然后从polα到polsδ或ε进行交接。我们的第二个项目解决了两个 关于复制叉遇到异常DNA的知识的关键差距。 RPA和RAD51是两个高度 最丰富的ssDNA结合蛋白在失速叉的失速，逆转和稳定中具有关键作用。 涂有RPA的ssDNA是多个损伤响应途径的关键启动信号，并播放几个 其他角色，包括招募和指导ATP运动蛋白Smarcal1的叉逆活性。 我们建议阐明通过确定的机制来驱动叉子重塑的这一重要方面 使用Cyro-Em，在模型叉子底物复合物上RPA和SMARCAL1的结构。 rad51扮演 在稳定的复制叉的稳定中的重要作用。与大卫·科尔特斯（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DNA损伤响应蛋白 停滞叉子的rad51。我们最近发现RADX还与RPA进行了物理相互作用，这表明有 RPA-RADX-RAD51网络在停滞的叉子上运行。我们提出结合结构，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在失速处的作用 复制叉。在一起，我们的两个项目将大大增强对DNA处理方式的理解 维持和传播真核复制叉和基因组。