MECHANISMS OF ESCHERICHIA COLI CHROMOSOMAL REPLICATION

大肠杆菌染色体复制机制

• 批准号: 6129338
• 负责人: JON M KAGUNI
• 金额: $ 27.36万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6129338
• 关键词: DNA binding protein; DNA footprinting; DNA replication; DNA replication origin; Escherichia coli; bacterial genetics; bacterial proteins; cell growth regulation; gene mutation; genetic mapping; laboratory rabbit; microorganism growth; molecular assembly /self assembly; protein protein interaction; protein sequence; site directed mutagenesis

The E. coli genome is a circular duplex DNA molecule containing a single replication origin, oriC. DNA replication that starts at this site is initiated by the binding of DnaA protein to specific sequences termed DnaA boxes. A series of discrete events follow to establish the protein machinery at each replication fork for bidirectional replication fork movement. This process to produce two progeny DNA molecules is coordinated to cell growth and is regulated at the step of initiation of chromosomal DNA replication. The long term objectives of this research are to understand the process of initiation of E. coli chromosomal DNA replication at the biochemical level, and to determine how this process is regulated. Our recent results indicate that DnaA protein interacts directly with DnaB in its entry at oriC. Furthermore, DnaA protein plays an active role in the loading of DnaB onto the DNA. In the next funding period, we will pursue several aims to establish how an intermediate of the initiation process termed the prepriming complex is assembled and how these proteins interact. Specific aims are: i) to determine the stoichiometry of DnaA, DnaB and DnaC in the prepriming complex formed at oriC and to investigate possible mechanisms that control the entry of DnaB, ii) to identify where DnaB enters at oriC, iii) to identify specific amino acids of DnaA that are important for binding to DnaB, iv) to determine of the physical form of DnaB protein that interacts with DnaA protein, v) to identify the regions of DnaA protein involved in self-aggregation, vi) to identify and characterize other proteins that bind to DnaA protein as an affinity ligand, and vii) to identify amino acids of DnaC that are important for interaction with DnaB. These studies will provide further insight on the role of DnaA protein in the initiation process. Furthermore, the biochemical events in the initiation process in this model system may have similarities to the mechanism of initiation of chromosomal replication in other organisms.

大肠杆菌基因组是一个环状的双链DNA分子，包含一个单一的复制起点ORIC。从这个位点开始的DNA复制是由Dna A蛋白与称为Dna A盒的特定序列结合而启动的。随后发生了一系列离散事件，从而在每个复制叉处建立了双向复制叉运动的蛋白质机制。这个产生两个后代DNA分子的过程是与细胞生长相协调的，并在启动染色体DNA复制的步骤中受到调节。这项研究的长期目标是在生化水平上了解大肠杆菌染色体DNA复制的启动过程，并确定这一过程是如何调控的。我们最近的研究结果表明，DNAA蛋白在进入ORIC时直接与DNAB相互作用。此外，DNAA蛋白在将DNAB装载到DNA中起着积极的作用。在下一个资助期，我们将追求几个目标，以确定称为预启动复合体的启动过程的中间体是如何组装的，以及这些蛋白质如何相互作用。具体目标是：i)确定Dna A、Dna B和Dna C在ORIC形成的预启动复合体中的化学计量比，并研究控制Dna B进入ORIC的可能机制，ii)确定Dna B进入ORIC的位置，iii)确定Dna A中对与Dna B结合很重要的特定氨基酸，iv)确定与Dna A蛋白相互作用的Dna B蛋白的物理形式，v)确定Dna A蛋白参与自我聚集的区域，vi)鉴定和表征作为亲和配体与Dna A蛋白结合的其他蛋白质，以及vii)确定Dna C中对与Dna B相互作用重要的氨基酸。这些研究将进一步深入了解DNAA蛋白在启动过程中的作用。此外，该模型系统中启动过程中的生化事件可能与其他生物中启动染色体复制的机制相似。