Structure and function of the bacterial primosome

细菌引发体的结构和功能

• 批准号: 8723244
• 负责人: James L Keck
• 金额: $ 28.38万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8723244
• 关键词: Bacteria; Binding; Biochemical; Biochemical Genetics; Biological; Cell physiology; Cells; Chromosome Mapping; Complex; DNA; DNA Polymerase I; DNA Structure; DNA biosynthesis; DNA replication fork; Data; Disease; DnaB helicase; Ensure; Genetic; Genome; Genomics; Goals; Hand; Health; Human; In Vitro; Individual; Knowledge; Length; Life; Link; Longevity; Maintenance; Malignant - descriptor; Maps; Measures; Mediating; Metabolic; Molecular; Molecular Models; Organism; Pathway interactions; Plague; Process; Protein Binding; Proteins; Publishing; Reaction; Regulation; Replication Initiation; Research; Resolution; Roentgen Rays; SS DNA BP; Site; Solutions; Structure; Surface; System; Testing; Translating; Variant; X-Ray Crystallography; base; emergency service responder; in vivo; killings; molecular modeling; physical model; prevent; programs; protein complex; protein function; recombinase; recombinational repair; repaired; research study; tumor

DESCRIPTION (provided by applicant): The DNA replication restart pathways reload the DNA replication machinery onto replication forks that have been abandoned as a consequence of genomic damage. These pathways form an essential biochemical link between repair (often recombinational repair) of broken replication forks and DNA replication. The proteins that drive these reactions, referred to as the primosome or the Replication Restart Proteins (RRPs), must recognize the structures of these abandoned replication forks and reload the DNA replication machinery at these sites. This process is heavily regulated to ensure loading fidelity and to avoid over-replication that could arise from initiating replication at improper DNA structures. The structural mechanisms underlying DNA replication restart and the cellular mechanisms by which it is integrated with other cellular genome maintenance processes are currently poorly understood. Our proposal combines structural, biochemical, and genetic approaches to define the mechanisms of DNA replication restart pathways in complementary ways. We wil use X- ray crystallography to determine the crystal structures of key proteins and protein complexes that comprise the primosome (Aim 1). These studies will produce molecular models that will help define the physical mechanisms by which bacterial RRPs function. Additionally, we will define biochemically how RRPs interact with one another to drive replication restart (Aim 2). This set of experiments will link the physical models generated in Aim 1 to steps along the replication restart pathways, to reveal how the primosome ties replication fork recognition to RRP complex assembly. Finally, we will identify linkages that coordinate replication restart with DNA replication, recombination, and repair processes in bacterial cells (Aim 3). These connections will help define how replication restart is integrated into the basal genome maintenance network in cells and how its use is regulated to prevent unwarranted replication initiation.

描述（由申请人提供）：DNA复制重启途径将DNA复制机器重新加载到因基因组损伤而被废弃的复制叉上。这些途径在断裂复制叉的修复（通常是重组修复）和 DNA 复制之间形成了重要的生化联系。驱动这些反应的蛋白质，称为引发体或复制重启蛋白质 (RRP)，必须识别这些废弃的复制叉的结构，并在这些位点重新加载 DNA 复制机器。该过程受到严格监管，以确保加载保真度并避免因在不正确的 DNA 结构处启动复制而可能出现的过度复制。这 目前，人们对 DNA 复制重启的结构机制及其与其他细胞基因组维护过程整合的细胞机制知之甚少。我们的建议结合了结构、生化和遗传学方法，以互补的方式定义 DNA 复制重启途径的机制。我们将使用 X 射线晶体学来确定构成引发体的关键蛋白质和蛋白质复合物的晶体结构（目标 1）。这些研究将产生分子模型，帮助定义细菌 RRP 发挥作用的物理机制。此外，我们将从生化角度定义 RRP 如何相互作用以驱动复制重新启动（目标 2）。这组实验将把目标 1 中生成的物理模型与复制重启路径的步骤联系起来，以揭示引发体如何将复制叉识别与 RRP 复合体组装联系起来。最后，我们将确定细菌细胞中协调复制重启与 DNA 复制、重组和修复过程的联系（目标 3）。这些连接将有助于定义如何将复制重启整合到细胞中的基础基因组维护网络中，以及如何调节其使用以防止不必要的复制启动。