MECHANISMS OF ESCHERICHIA COLI CHROMOSOMAL REPLICATION

描述（改编自研究者摘要）：染色体 大肠杆菌的复制受到调控，并与 细菌细胞周期 这项研究的长期目标是 从生物化学上理解染色体复制的起始， 其规定。 在参与免疫反应的众多蛋白质中， 在DNA复制过程中，DnaA蛋白由466个氨基酸残基组成， 在启动过程中的特殊作用是独一无二的。 作为一个序列- 特异性DNA结合蛋白，它结合到染色体上的位点 origin，oriC，染色体复制开始的位点。 对 绑定时，它会介导一系列事件， 启动DNA复制。 通过一种新的遗传方法， 分离并表征。 我们希望能找出 DnaA蛋白在DNA复制中的活性。 我们将继续遗传 和这些新的dnaA突变体的生物化学表征。 三 在这个总目标下提出了四个目标。 第一个具体目标 目的是鉴定DnaA蛋白的DNA结合结构域， 参与这项活动。第二个目标是确定函数 同源物中高度保守序列P-环基序的意义 也存在于许多核苷酸结合蛋白中。生化 分离的P-环突变体的表征， 这些突变体在宿主中具有显性负效应， dnaA+等位基因、P环诱变和交联研究 被提议。 此外，突变体的集合可能包含那些 除了DNA结合和ATP之外， 约束力 作为第三个目标，对这些突变体进行生物化学研究， 提出了 这些研究将有望提供更多的洞察力， DnaA蛋白在启动过程中的作用。 作为第四个目标， 我们计划鉴定出与DnaA蛋白质发生物理相互作用的蛋白质， 通过其作为亲和配体的用途。 这些研究的结果涉及 复制设备如何在E.杆菌 染色体起源，以及其他蛋白质是否在物理上相互作用 调节DnaA蛋白的活性。 这一目标包括一项研究， 为了确定DnaA、DnaB和DnaC蛋白质的化学计量， 分离的预引发复合物。 总的来说，这些研究承诺 进一步了解DnaA蛋白在起始过程中的作用， 并提供一个模型系统，由此该过程可以在其他系统中发生， 有机体

DESCRIPTION (adapted from investigator's abstract): Chromosomal replication of Escherichia coli is regulated and coordinated to the bacterial cell cycle. The long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. Among the numerous proteins that are involved in the process of DNA replication, DnaA protein of 466 amino acid residues is unique in its specific role in the initiation process. As a sequence- specific DNA binding protein, it binds to sites in the chromosomal origin, oriC, the site where chromosomal replication begins. On binding, it then mediates a series of events that establishes the initiation of DNA replication. By a novel genetic method, a large collection of mutants has been isolated and characterized. We hope to identify residues essential for activity of DnaA protein in DNA replication. We will continue genetic and biochemical characterization of these novel dnaA mutants. Three of four aims are proposed under this general goal. The first specific aim is to identify the DNA binding domain of DnaA protein, and residues involved in this activity. The second aim is to determine the functional significance of a P-loop motif, a sequence highly conserved in homologs of dnaA and also found in many nucleotide binding proteins. Biochemical characterization of isolated P-loop mutants, investigation of the dominant-negative effect these mutants have when in a host that contains the dnaA+ allele, mutagenesis of the P-loop, and crosslinking studies are proposed. In addition, the collection of mutants may contain those that are defective in essential functions other than DNA binding and ATP binding. As a third aim, a biochemical study of these mutants is proposed. These studies will hopefully provide more insight into the function of DnaA protein in the initiation process. As a fourth aim, we plan to identify proteins that interact physically with DnaA protein by its use as an affinity ligand. Findings from these studies address the issue of how the replication apparatus assembles at the E. coli chromosomal origin, and whether other proteins interact physically to modulate the activity of DnaA protein. Included in this aim is a study to determine the stoichiometry of DnaA, DnaB, and DnaC proteins in isolated prepriming complexes. Collectively, these studies promise further insight on the role of DnaA protein in the initiation process, and provide a model system whereby this process may occur in other organisms.