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复制重新启动途径将DNA复制机械重新加载到复制叉上，这些复制叉因基因组损伤而被放弃。这些途径在破裂的复制叉和DNA复制之间的修复（通常是重组修复）之间形成了必不可少的生化联系。驱动这些反应的蛋白质，称为原始体或复制重新启动蛋白质（RRP），必须识别这些废弃复制叉的结构，并在这些位点重新加载DNA复制机制。该过程受到严格的调节，以确保加载保真度并避免因在不当DNA结构上启动复制而产生的过度复制。这 目前对DNA复制重新启动的结构机制及其与其他细胞基因组维持过程集成的细胞机制目前尚不清楚。我们的建议结合了结构，生化和遗传方法，以定义DNA复制的机制以互补的方式重新启动途径。我们将使用X-Ray晶体学来确定包含原始体的关键蛋白质和蛋白质复合物的晶体结构（AIM 1）。这些研究将产生分子模型，以帮助定义细菌RRP功能的物理机制。此外，我们将在生化上定义RRP如何相互作用以驱动复制重新启动（AIM 2）。这组实验将将AIM 1中生成的物理模型与沿复制重新启动途径的步骤联系起来，以揭示原始纽带复制叉的识别如何与RRP复杂组件。最后，我们将确定与细菌细胞中的DNA复制，重组和修复过程重新启动的链接（AIM 3）。这些连接将有助于定义如何将复制重新启动集成到单元中的基础基因组维持网络中，以及如何调节其使用以防止不必要的复制启动。