MECHANISMS OF ESCHERICHIA COLI CHROMOSOMAL REPLICATION

急诊大肠杆菌基因组是含有单一复制起点oriC的环状双链DNA分子。在该位点开始的DNA复制通过DnaA蛋白与称为DnaA盒的特定序列结合而启动。一系列离散事件随后在每个复制叉处建立蛋白质机制以进行双向复制叉运动。这种产生两个子代DNA分子的过程与细胞生长协调，并在染色体DNA复制起始的步骤中受到调节。本研究的长期目标是了解E.大肠杆菌染色体DNA复制的生化水平，并确定如何这一过程是受管制的。我们最近的研究结果表明，DnaA蛋白直接与DnaB在其进入oriC。此外，DnaA蛋白在将DnaB装载到DNA上方面起着积极的作用。在下一个资助期内，我们将追求几个目标，以建立一个中间的启动过程称为prepriming复合物是如何组装和这些蛋白质如何相互作用。具体目标是：i）确定在oriC处形成的预引发复合物中DnaA、DnaB和DnaC的化学计量，并研究控制DnaB进入的可能机制，ii）鉴定DnaB在oriC处进入的位置，iii）鉴定DnaA中对结合DnaB重要的特定氨基酸，iv）确定与DnaA蛋白相互作用的DnaB蛋白的物理形式，v）鉴定参与自聚集的DnaA蛋白的区域，vi）鉴定和表征作为亲和配体与DnaA蛋白结合的其它蛋白，和vii）鉴定对于与DnaB相互作用重要的DnaC的氨基酸。这些研究将为DnaA蛋白在启动过程中的作用提供进一步的见解。此外，在该模型系统中的起始过程中的生化事件可能与其他生物体中染色体复制起始的机制具有相似性。

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.