Structural Biology of Multi-Domain Proteins and Multi-Protein Machinery in DNA Replication and Repair

DNA 复制和修复中多域蛋白和多蛋白机制的结构生物学

• 批准号: 10393403
• 负责人: WALTER J. CHAZIN
• 金额: $ 1.26万
• 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 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393403
• 关键词: Address; Amino Acid Sequence; Architecture; Bacteria; Biochemical; Biological; Biological Models; Cells; Complement; Complex; DNA; DNA Damage; DNA Primase; DNA Primers; DNA Repair; DNA Replication Damage; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Defect; Development; Disease; Genome; Genomic Instability; Goals; Hand; Human; Knowledge; Lead; Length; Lower Organism; Maintenance; Malignant Neoplasms; Modernization; Multiprotein Complexes; Mutation; Patients; Phase; Polymerase; Process; Proteins; RNA; Replication Origin; Research; Role; SS DNA BP; Signaling Protein; Simian virus 40; Single-Stranded DNA; Structural Protein; Structure; Tertiary Protein Structure; Validation; base; chemotherapy; helicase; human DNA; mutant; protein structure; recruit; replication factor A; response; structural biology; targeted treatment; three dimensional structure; tool

PROJECT SUMMARY Faithful replication of DNA and response to damaged DNA, essential to cell propagation and survival, proceeds via the action of multi-protein machines. While considerable progress has been made in elucidating the mechanisms of DNA replication and damage response from studies of bacteria and other lower organisms, information on humans is lacking because the protein sequences and structures are not conserved. Our long- term goals are to understand the action of human DNA replication and damage response machinery. Our strategy is to elucidate the structural basis for these processes using the full complement of modern structural biology tools and interpret this information using a range of biochemical and biological approaches. We have shown in the SV40 model system that the helicase that creates the single-stranded DNA (ssDNA) at the origin of replication also actively loads the human ssDNA binding protein replication protein A (RPA), and that this action is essential for DNA priming by DNA polymerase -primase (pol-prim). We have also characterized the complex and dynamic structural architectural of the modular RPA protein as it engages this ssDNA template and have begun to address the quandary of how RPA releases the template to hand off to pol-prim. Once the primase subunits are loaded on the DNA template, an ~10 nt RNA is synthesized and the primed template is transferred to the pol  subunits for extension to the final ~30 nt of RNA-DNA primer. Our goal is to continue to define the trajectory of actions on the template as it is replicated. The next phase of our research is to solve the fundamental mysteries about the release of RPA and loading of pol-prim onto the template, the counting of primer length by primase, and the hand-offs from primase to pol  and in turn to pol  or pol . Our DNA damage response research seeks to understand the mechanism of action of the key proteins involved, discern the biochemical bases for malfunctions caused by disease-associated mutations, and ultimately use this knowledge to help evaluate the potential of chemotherapies targeted to these proteins. Here again, RPA has a critical role, in this case, in recruiting partner proteins that stall and remodel replication forks and activate important signaling proteins. The goal is to obtain structural understanding and functional validation of the interactions between RPA and damage response proteins, and their role in responding to encounters with damaged DNA.

项目摘要 DNA的忠实复制和对受损的DNA的反应，对细胞的传播和生存至关重要，继续进行 通过多蛋白机的作用。虽然在阐明 DNA复制的机制和细菌和其他较低生物的研究的损伤反应， 缺乏有关人类的信息，因为蛋白质序列和结构不能保守。我们的长期 术语目标是了解人类DNA复制和损坏响应机械的作用。我们的 策略是使用现代结构的完整完成来阐明这些过程的结构基础 生物学工具并使用一系列生化和生物学方法来解释此信息。我们有 在SV40模型系统中显示的是在原点创建单链DNA（ssDNA）的解旋酶 复制还积极地加载了人类ssDNA结合蛋白复制蛋白A（RPA），并且 动作对于通过DNA聚合酶-原酶（POL-PRIM）的DNA启动至关重要。我们还表征了 模块化RPA蛋白的复杂而动态的结构架构与此SSDNA模板和 已经开始解决RPA如何释放模板以将模板移交给POL-PRIM的惯例。一旦 原始子亚基被加载在DNA模板上，合成了一个约10 nt RNA，并且引发模板为 转移到pol的亚基中，以扩展到RNA-DNA底漆的最终〜30 nt。我们的目标是继续 在复制模板上定义动作的轨迹。我们研究的下一个阶段是解决 关于释放RPA和将POL-PRIM加载到模板上的基本奥秘，计数 底漆长度的底漆，以及从原始酶到pol的交接，然后又转到pol或pol。我们的DNA损伤 反应研究旨在了解所涉及的关键蛋白质的作用机理，辨别 由疾病相关突变引起的出现故障的生化基础，并最终使用这些知识 帮助评估针对这些蛋白质的化学疗法的潜力。再次，RPA具有关键作用， 在这种情况下，在招募合作伙伴蛋白质中，这些蛋白会停滞和重塑叉并激活重要的信号传导 蛋白质。目的是获得对相互作用的结构理解和功能验证 RPA和损伤反应蛋白，以及它们在响应损坏DNA的相遇中的作用。