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

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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.