Biochemical and genetic study of the regulatory mechanism of the chromosomal DNA replication in Escherichia coli

Dna A蛋白是大肠杆菌中染色体DNA复制的启动子。首席研究员和同事们表明，Dna A蛋白可被包括心磷脂在内的酸性磷脂激活。在这项研究中，我们研究了由两种不同合成脂类组成的混合脂质体对DNAA蛋白结合ATP的影响。结果表明，混合膜中酸性脂的团簇结构是酸性脂与DNAA蛋白相互作用的必要条件。这一发现为细胞分裂周期中DNA复制的起始蛋白Dna A蛋白的活性调控提供了新的见解。也就是说，酸性磷脂可以在细胞膜上形成分散的结构簇，脂结构的结构变化可能与细胞周期相耦合，激活Dna A蛋白，从而启动DNA复制。此外，我们首次报道了Dna A蛋白对双链DNA超螺旋结构的影响。我们发现，当DNA拓扑异构酶在Dna A蛋白存在的情况下进行反应时，DNA更负向超螺旋。DNAA蛋白的这种活性不依赖于DNA序列，并被DNAA蛋白与腺核苷酸的结合特异性地抑制。

DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. The head investigator and colleagues showed that DnaA protein is activated by acidic phospholipids including cardiolipin. In this study, we examined the influcnce of mixed liposomes composed of two different synthetic lipids on the ATP-binding of DnaA protein. The result showed that the cluster structure of acidic lipid is necessary for the interaction of acidic lipid in the mixed membrane with DnaA protein. This finding suggests a new insight for the regulation of the activity of DnaA protein, the initiator protein of DNA replication, in the cycle of cell division. Namely, acidic phospholipids may form either the cluster of dispersd structures in cell membrane, and that structural alternation of lipid structure may be coupled with cell cycle and it activates DnaA protein resulting the initiation of DNA replication.Further, we first reported the influence of DnaA protein on the supercoiled structure of double stranded DNA.We showed that DNA is more negatively supercoiled when the reaction of DNA topoisomerase was carried out in the presence of DnaA protein. This activity of DnaA protein was not dependent on sequence of DNA and was specifically inhibited by binding of DnaA protein to adenine nucleotide.