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

本研究旨在了解大肠杆菌DNA复制起始因子DnaA蛋白的调控机制。首席研究员等人先前报道了（i）DnaA蛋白对ATP具有高亲和力，（ii）与DnaA蛋白结合的ATP被水解以形成ADP结合形式的DnaA蛋白，其对DNA复制无活性，和（iii）DNA聚合酶III全酶，即大肠杆菌中的复制酶，刺激与DnaA蛋白结合的ATP的水解。本研究建立了一种新的体内测定DnaA蛋白结合腺嘌呤核苷酸的方法。用[^<32>P]正磷酸盐进行放射性标记的细胞均质化，通过免疫沉淀回收DnaA蛋白，然后通过薄层色谱法进行分析。通过使用各种温度敏感的DNA复制突变体，我们证明了负责形成复制叉的复制蛋白参与与起始蛋白结合的水解ATP的反应。我们还建立了一种方法来测定磷脂结合DnaA蛋白。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。我们检测到心磷脂和磷脂酰甘油，这是以前显示结合DnaA蛋白在体外，在免疫沉淀。结果表明DnaA蛋白与细胞内的酸性磷脂结合。

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.