Cytology and function of ParA in E. coli

大肠杆菌中 ParA 的细胞学和功能

• 批准号: 7939920
• 负责人: JOSEPH A POGLIANO
• 金额: $ 19.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7939920
• 关键词: ATP phosphohydrolase; Address; Bacteria; Behavior; Binding; Biological; Cells; Chromosome Segregation; Chromosomes; Complex; Coupled; Cytology; Cytoskeletal Proteins; DNA; DNA Sequence; DNA-Binding Proteins; Electrons; Escherichia coli; Exhibits; F Factor; Fluorescence; Fluorescence Microscopy; In Vitro; Laboratories; Mediating; Microscopy; Nucleoproteins; Plasmids; Play; Polymers; Process; Protein Family; Proteins; Recovery; Reporting; Role; Site; System; Time; depolymerization; fluorescence microscope; in vivo; plasmid DNA; polymerization; segregation; time use

ParA systems play an important role in chromosome segregation and are responsible for the segregation of many bacterial plasmids. This proposal focuses on the ParA partitioning system of E. coli plasmid F, which consists of the SopA (ParA) ATPase, the SopB (ParB) DNA binding protein, and a DNA sequence, sopC, which contains the recognition sites for SopB binding. Our cell biological studies have demonstrated that SopA-GFP assembles into dynamic polymers that appear in the fluorescence microscope as a bright cloud surrounding the plasmid. This dynamic assembly of SopA is regulated by the SopB/sopC nucleoprotein complex in vivo and is essential for the ability of the Sop system to perform DNA segregation. The mechanism by which a ParA system performs DNA segregation remains unknown. We do not yet understand the mechanism of SopA polymerization or how polymerization contributes to each of the steps in plasmid segregation. We therefore propose to investigate the mechanism of ParA- mediated plasmid segregation in order to gain a better understanding of how this widespread family of proteins contributes to DNA segregation in bacteria. Specifically, we will characterize SopA polymerization in vivo using fluorescence recovery after photopbleaching (FRAP), total internal reflection fluorescence (TIRF) microscopy and speckle microscopy. Using time-lapse fluorescence microscopy and electron cryotomography, we will characterize the dynamic behavior of SopA during oscillation and plasmid separation. These studies will determine some of the basic features of SopA in vivo polymerization behavior and determine how polymerization is coupled to DNA segregation. The findings from this analysis of a representative ParAB plasmid partitioning system should have wide implications for our understanding of plasmid and chromosome segregation.

ParA 系统在染色体分离中发挥着重要作用，负责 许多细菌质粒的分离。本提案重点关注ParA 大肠杆菌质粒 F 的分配系统，由 SopA (ParA) ATPase、 SopB (ParB) DNA 结合蛋白和 DNA 序列 sopC，其中包含 SopB 结合的识别位点。我们的细胞生物学研究表明 SopA-GFP 组装成荧光显微镜中出现的动态聚合物 作为质粒周围的明亮云。 SopA 的这种动态组装受到调控 通过体内 SopB/sopC 核蛋白复合物，对于 Sop系统进行DNA分离。 ParA 系统的机制 执行 DNA 分离仍然未知。我们还不了解其中的机制 SopA 聚合的原理或聚合如何对每个步骤做出贡献 质粒分离。因此，我们建议研究ParA-的机制 介导的质粒分离，以便更好地了解这是如何进行的 广泛的蛋白质家族有助于细菌中的 DNA 分离。具体来说， 我们将使用荧光恢复来表征 SopA 体内聚合 光漂白 (FRAP)、全内反射荧光 (TIRF) 显微镜和 散斑显微镜。使用延时荧光显微镜和电子 冷冻断层扫描，我们将表征 SopA 在振荡过程中的动态行为 和质粒分离。这些研究将确定 SopA 的一些基本特征 体内聚合行为并确定聚合如何与 DNA 偶联 隔离。对代表性 ParAB 质粒的分析结果 分配系统应该对我们对质粒和质粒的理解产生广泛的影响。 染色体分离。