MECHANISMS OF ESCHERICHIA COLI CHROMOSOMAL REPLICATION

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。DNA复制开始于这个位点，是由DNA蛋白与称为DNA盒子的特定序列结合而开始的。一系列离散事件随后在每个复制叉建立双向复制叉运动的蛋白质机制。这一产生两个子代DNA分子的过程与细胞生长相协调，并在染色体DNA复制起始阶段受到调控。本研究的长期目标是在生化水平上了解大肠杆菌染色体DNA复制的起始过程，并确定这一过程是如何被调节的。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，DNA蛋白在DNA的装载过程中起着积极的作用。在下一个资助期，我们将追求几个目标，以确定启动过程的中间产物如何组装，以及这些蛋白质如何相互作用。具体目标是：i)确定在oriC形成的预引复合体中DnaA、DnaB和DnaC的化学计量，并研究控制DnaB进入的可能机制，ii)确定DnaB进入oriC的位置，iii)确定与DnaB结合重要的DnaA特定氨基酸，iv)确定与DnaA蛋白相互作用的DnaB蛋白的物理形式，v)确定参与自聚集的DnaA蛋白区域。vi)鉴定和表征与DnaA蛋白结合为亲和配体的其他蛋白质，以及vii)鉴定与DnaB相互作用重要的DnaC氨基酸。这些研究将进一步揭示dna蛋白在起始过程中的作用。此外，该模型系统启动过程中的生化事件可能与其他生物体中染色体复制的启动机制有相似之处。

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.