MECHANISMS OF ESCHERICHIA COLI CHROMOSOMAL REPLICATION

描述(改编自研究人员摘要)：染色体 大肠埃希氏菌的复制受到调控和协调 细菌细胞周期。这项研究的长期目标是 从生物化学角度了解染色体复制的启动，以及 它的规定。在众多的蛋白质中，参与了 在DNA复制过程中，DNAA蛋白的466个氨基酸残基是 其独特之处在于其在启动过程中的具体作用。作为一个序列- 特定的DNA结合蛋白，它结合到染色体上的位置 起源，ORIC，染色体复制开始的地方。在……上面 绑定，然后它调解一系列事件，这些事件建立 启动DNA复制。 通过一种新的遗传方法，一大批突变体被 与世隔绝的，有特点的。我们希望能识别出对 DNA复制过程中DNAA蛋白的活性我们将继续遗传 并对这些新的DNAA突变体进行了生化特征分析。其中三个 在这一总目标下提出了四个目标。第一个具体目标 是鉴定DNAA蛋白的DNA结合域，以及残基 参与了这项活动。第二个目标是确定功能 同源基因中高度保守的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-环突变体的特性，对 这些突变体在含有以下物质的宿主中具有显性-负效应 DNAA等位基因、P-环的突变和交联性研究 都被提出了。此外，突变体的集合可能包含那些 在DNA结合和ATP以外的基本功能上有缺陷 有约束力的。作为第三个目标，对这些突变体的生化研究是 建议。这些研究有望为我们提供更多关于 Dna A蛋白在启动过程中的作用。作为第四个目标， 我们计划鉴定与DNAA蛋白发生物理作用的蛋白质 通过将其用作亲和配体。这些研究的发现解决了 复制装置如何在大肠杆菌中组装的问题 染色体起源，以及其他蛋白质是否物理上相互作用 调节DNAA蛋白的活性。这一目标包括一项研究 测定DNAA、DNAB和DNAC蛋白的化学计量比 孤立的预引爆复合体。总而言之，这些研究承诺 进一步了解DNAA蛋白在启动过程中的作用， 并提供一个模型系统，通过该模型系统，该过程可以在其他 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.