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

DnaA蛋白是大肠杆菌染色体DNA复制的启动子。首席研究员和同事们表明，DnaA蛋白被酸性磷脂（包括心磷脂）激活。本文研究了由两种不同合成脂质组成的混合脂质体对DnaA蛋白ATP结合的影响。结果表明，混合膜中酸性脂质的簇结构是酸性脂质与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复制起始蛋白DnaA蛋白活性的调节提供了新的见解。也就是说，酸性磷脂可以在细胞膜中形成分散结构的簇，并且脂质结构的结构改变可以与细胞周期偶联，并且它激活DnaA蛋白，导致DNA复制的启动。我们首次报道了DnaA蛋白对双链DNA超螺旋结构的影响，发现当DNA拓扑异构酶的反应为在DnaA蛋白存在下进行。DnaA蛋白的这种活性不依赖于DNA的序列，并通过与腺嘌呤核苷酸的结合而被特异性抑制。

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.