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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中DNA复制的启动子Dna A蛋白的调控机制。首席调查员等人。先前报道：(I)Dna A蛋白与ATP有很高的亲和力，(Ii)与Dna A蛋白结合的ATP被水解形成Dna A蛋白的ADP结合形式，该形式对DNA复制不起作用，以及(Iii)DNA聚合酶III全酶，即在大肠杆菌中的复制酶，刺激与Dna A蛋白结合的ATP的水解性。在本研究中，我们建立了一种在体内测定与DNAA蛋白结合的腺嘌呤核苷酸的新方法。[^&lt；32&gt；P]正磷酸盐标记细胞匀浆，免疫沉淀法回收DNAA蛋白，薄层层析分析。通过使用各种温度敏感的DNA复制突变体，我们证明了负责复制叉形成的复制蛋白参与了与启动蛋白结合的水解性ATP的反应。我们还建立了一种测定DNAA蛋白结合磷脂的方法。用有机溶剂提取放射性标记免疫沉淀物，用薄层层析进行分析。我们在免疫沉淀物中检测到心磷脂和磷脂酰甘油，这两种物质在体外被证明与DNAA蛋白结合。结果表明，Dna A蛋白与细胞内的酸性磷脂结合。