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: 10382072
• 负责人: WALTER J. CHAZIN
• 金额: $ 3.7万
• 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/10382072
• 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DNA的反应，对细胞繁殖和存活至关重要， 通过多蛋白质机器的作用。虽然在阐明 从细菌和其他低等生物的研究中发现的DNA复制和损伤反应机制， 由于蛋白质序列和结构不保守，因此缺乏关于人类的信息。我们长久以来- 本学期的目标是了解人类DNA复制和损伤反应机制的作用。我们 战略是阐明这些过程的结构基础，使用现代结构的完整补充， 生物学工具，并使用一系列生物化学和生物学方法解释这些信息。我们有 在SV 40模型系统中显示，在起始处产生单链DNA（ssDNA）的解旋酶 也主动加载人ssDNA结合蛋白复制蛋白A（RPA），这是一个非常重要的发现。 作用对于DNA聚合酶β-引发酶（pol-primase）的DNA引发是必不可少的。我们还描述了 模块化RPA蛋白的复杂和动态的结构架构，因为它接合该ssDNA模板， 已经开始解决RPA如何发布模板以移交给Pol-Cash的难题。一旦 将引物酶亚基加载到DNA模板上，合成约10 nt的RNA，并将引物模板 转移到聚合酶亚基上，延伸到RNA-DNA引物的最后~30 nt。我们的目标是继续 在复制模板时定义模板上的操作轨迹。我们研究的下一阶段是解决 关于RPA的释放和模板上的聚合物的加载的基本奥秘， 引物长度由引物酶决定，以及从引物酶到聚合酶，再到聚合酶或聚合酶的传递。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制， 疾病相关突变引起的功能障碍的生物化学基础，并最终使用这些知识 以帮助评估针对这些蛋白质的化疗的潜力。在这方面，爱国军再次发挥关键作用， 在这种情况下，在招募伴侣蛋白，停止和重塑复制叉和激活重要的信号 proteins.目的是获得结构的理解和功能验证之间的相互作用 RPA和损伤反应蛋白，以及它们在应对受损DNA时的作用。