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

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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.