Study of the mechanism of bacterial chromosome partitioning systems

细菌染色体分配系统机制研究

• 批准号: 7967404
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 31.28万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967404
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Anti-Bacterial Agents; Bacterial Chromosomes; Binding; Biochemical; Cell division; Cells; Centromere; Chromosomes; DNA; DNA Binding; DNA Sequence; DNA biosynthesis; Daughter; Detection; Development; Dissociation; Ensure; Escherichia coli; F Factor; Fibrinogen; Fluorescence; Fluorescent Dyes; Foundations; Genome; Glass; Goals; Image; Inherited; Kinetics; Label; Learning; Modeling; Molecular; Monitor; Movement; Plasmids; Process; Protein Binding; Protein Dynamics; Proteins; Reaction; Slide; Surface; System; Techniques; Transport Process; Transport Reaction; Transportation; charge coupled device camera; chromosome loss; daughter cell; fluorescence microscope; in vivo; instrument; novel; nucleotide analog; plasmid DNA; single molecule

After DNA replication, two daughter copies of bacterial chromosomes or low copy number plasmids must be segregated into two daughter cells in order to avoid rapid chromosome loss. Therefore, systems have evolved to actively partition the replicated copies of the genome to two halves of the cell before cell division takes place. One class of such systems involve a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of centromere, and two protein factors, one binds to the centromere and the other an ATPase with non-specific DNA binding activity. E. coli P1 plasmid and F plasmid are both equipped with such systems. The centromere of P1 plasmid is called ParS, to which ParB protein binds, and ParA is the ATPase. The centromere of F plasmid is called SopC, to which SopB protein binds, and SopA is the ATPase. In vivo imaging studies on some of these systems have demonstrated oscillating focus formation of the ATPase protein and accompanied oscillation of the plasmid DNA within the cell prior to DNA replication. After replication, one DNA copy stays near one end of the cell and the other copy moves toward the other end prior to cell division. However, detailed molecular mechanisms of these bio-molecular transport reaction systems are still poorly understood, due in part to the absence of suitable cell free reaction system to detect the DNA movements. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of this reaction system by combining a variety of techniques. Techniques and instruments have been developed to study these reactions at the single molecule detection level by using a sensitive fluorescence microscope/CCD camera system. Using GFP-tagged ParA and fluorescent dye-coupled ParB proteins, association/dissociation dynamics of these proteins with DNA molecules immobilized on a slide glass surface was monitored under a variety of reaction conditions. We learned that: ParA, in the presence of ATP associates with non-specific DNA with rapid on- and off-rates. No other nucleotide analogues have been found to be able to substitute ATP for ParA-DNA association. ParA conformational change induced by ATP binding has been observed. Pre-steady state kinetic analysis of the ParA ATPase reaction and the ATP-induced conformational change of ParA have been studied. The ATP-induced ParA conformational change, which could take place prior to ATP hydrolysis is required for ParA DNA binding. These observations will be combined to formulate a model for the mechanism of action of this reaction system. We also are in the process of initiating a parallel study of the F plasmid partitioning reaction. Fluorescence-labeled SopA and SopB proteins have been constructed and purified in active forms. The reaction system studied here is an example of a novel biomolecular transport reaction, and the experimental techniques developed here will be exploited for the parallel studies of mechanistically related reaction systems.

DNA复制后，细菌染色体或低拷贝质粒的两个子拷贝必须分离到两个子细胞中，以避免染色体快速丢失。 因此，系统已经进化到在细胞分裂发生之前主动地将基因组的复制拷贝分配到细胞的两半。 一类这样的系统涉及分离染色体上的特异性DNA序列，其作为着丝粒的细菌等同物起作用，以及两种蛋白质因子，一种结合着丝粒，另一种是具有非特异性DNA结合活性的ATP酶。 E. coli P1质粒和F质粒都配备了这样的系统。 P1质粒的着丝粒称为ParS，与ParB蛋白结合，帕拉是ATP酶。F质粒的着丝粒称为SopC，SopB蛋白与之结合，SopA是ATP酶。 对这些系统中的一些系统的体内成像研究已经证明了ATP酶蛋白的振荡焦点形成和伴随的DNA复制前细胞内质粒DNA的振荡。 复制后，一个DNA拷贝留在细胞的一端附近，另一个拷贝在细胞分裂前向另一端移动。然而，这些生物分子转运反应系统的详细分子机制仍然知之甚少，部分原因是缺乏合适的无细胞反应系统来检测DNA运动。 本项目旨在通过结合多种技术来研究该反应系统动力学方面的生物化学和生物物理机制。 技术和仪器已经开发，通过使用灵敏的荧光显微镜/CCD相机系统在单分子检测水平上研究这些反应。 使用GFP标记的帕拉和荧光染料偶联的ParB蛋白质，这些蛋白质与固定在载玻片表面上的DNA分子的缔合/解离动力学在各种反应条件下进行监测。我们了解到：帕拉，在ATP的存在下与非特异性DNA以快速的结合和解离速率相关联。 没有发现其他核苷酸类似物能够取代ATP的ParA-DNA协会。 观察到ATP结合引起的帕拉构象变化。 对帕拉ATP酶反应的预稳态动力学分析和ATP诱导的帕拉构象变化进行了研究。 ATP诱导的帕拉构象变化可能发生在ATP水解之前，是帕拉DNA结合所必需的。 这些观察结果将结合起来，制定一个模型，该反应系统的作用机制。 我们也在启动一个平行的研究过程中的F质粒分配反应。荧光标记的SopA和SopB蛋白已被构建并纯化为活性形式。 这里研究的反应系统是一个新的生物分子运输反应的例子，这里开发的实验技术将被利用的机械相关的反应系统的平行研究。