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起着重要作用，并负责 许多细菌质粒的分离。这项提案的重点是第#段 由SOPA(对位)ATPase组成的大肠杆菌质粒F的分区系统， SopB(PARB)DNA结合蛋白和DNA序列sopC，它包含 SopB结合的识别位点。我们的细胞生物学研究证明 SOPA-GFP组装成动态聚合物，出现在荧光显微镜下 就像环绕在质粒周围的一团明亮的云。SOPA的这种动态组装是受监管的 通过SopB/SopC核蛋白复合体在体内，并对 SOP系统进行DNA分离。一种辅助制度建立的机制 是否进行DNA分离仍不清楚。我们还不了解这种机制。 SOPA聚合的作用，或聚合作用如何影响 质粒分离。因此，我们建议研究副- 为了更好地理解这是如何进行的 广泛存在的蛋白质家族有助于细菌中的DNA分离。具体来说， 我们将在体内使用荧光恢复来表征SOPA聚合 光漂白(FRAP)、全内反射荧光(TIRF)显微镜和 散斑显微镜。使用时间推移荧光显微镜和电子 低温层析成像，我们将描述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 种族隔离。对一个具有代表性的Parab质粒的分析结果 分割系统应该对我们理解质粒和蛋白质有广泛的意义 染色体分离。