Study of the regulatory mechanism of DnaA protein, the initiator of DNA replication in Escherichia coli.

大肠杆菌DNA复制启动子DnaA蛋白调控机制的研究。

基本信息

批准号：
11480202
负责人：
SEKIMIZU Kazuhisa
金额：
$ 9.79万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480202/
关键词：
Escherichia coli initiation of DNA replication DnaA ATP ATPase RIDA replication fork phospholipid DnaA蛋白質 DNA複製複製開始制御 DNAポリメラーゼ抗菌剤

项目摘要

The purpose of this study is to understand the regulatory mechanism of DnaA protein, the initiator of DNA replication in Escherichia coli. The head investigator et al. previously reported that (i) DnaA protein has a high affinity for ATP, (ii) ATP bound to DnaA protein is hydrolyzed to form the ADP-binding form of DnaA protein, which is inactive for DNA replication, and, (iii) DNA polymerase III holoenzyme, the replicase in Escherichia coli, stimulates the hydrolysis of ATP bound to DnaA protein. In this study, we established a new method to determine adenine nucleotide bound to DnaA protein in vivo. Radio labeled cells with [^<32>P] orthophosphate were homogenized, and DnaA protein was recovered by immuno-precipitation, followed by analysis by thin layer chromatography. By using various temperature-sensitive mutants of DNA replication, we demonstrated that replication proteins responsible for the formation of the replication fork participate in the reaction of the hydrolysis ATP bound to the initiator protein. We also established a method to determine phospholipid bound to DnaA protein. The radiolabeled immuno-precipitates were extracted with organic solvents, and analyzed by thin layer chromatography. We detected cardiolipin and phosphatidylglycerol, which are previously shown to bind to DnaA protein in vitro, in the immuno-precipitates. The result demonstrates that DnaA protein binds to acidic phospholipids in cells.

本研究的目的是了解大肠杆菌 DNA 复制的启动子 DnaA 蛋白的调控机制。首席调查员等人。先前报道，(i) DnaA 蛋白对 ATP 具有高亲和力，(ii) 与 DnaA 蛋白结合的 ATP 被水解形成 DnaA 蛋白的 ADP 结合形式，该形式对 DNA 复制无活性，以及 (iii) DNA 聚合酶III 全酶是大肠杆菌中的复制酶，可刺激与 DnaA 蛋白结合的 ATP 的水解。在本研究中，我们建立了一种新方法来测定体内与 DnaA 蛋白结合的腺嘌呤核苷酸。将用[ 32 P]正磷酸盐放射性标记的细胞均质化，并通过免疫沉淀回收DnaA蛋白，随后通过薄层色谱法进行分析。通过使用DNA复制的各种温度敏感突变体，我们证明了负责形成复制叉的复制蛋白参与了与起始蛋白结合的水解ATP的反应。我们还建立了一种测定与 DnaA 蛋白结合的磷脂的方法。用有机溶剂提取放射性标记的免疫沉淀物，并通过薄层色谱法进行分析。我们在免疫沉淀物中检测到心磷脂和磷脂酰甘油，此前已证明它们在体外可与 DnaA 蛋白结合。结果表明 DnaA 蛋白与细胞中的酸性磷脂结合。