REGULATORY MACHINERY FOR THE E. COLI CHROMOSOMAL REPLICATION CYCLE

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In Escherichia coli, ATP-bound DnaA protein forms initiation complex with the replicational origin for chromosomal replication. I found that hydrolysis of DnaA-bound ATP is promoted by a specific DNA-protein complex called the sliding clamp which is formed upon loading of the DNA polymerase III (the chromosomal replicase) onto DNA. The resultant ADP-DnaA is inactive for initiation, and thus overinitiation is repressed (Cell, 1998; EMBO J., 1999). In this research program, I identified a novel clue for this system termed RIDA (regulatory inactivation of DnaA). This clue termed IdaB/Hda protein is required for RIDA in vitro and in vivo. Using purified IdaB/Hda protein, the sliding clamp, and DnaA protein, I succeeded construction of in vitro reconstituted RIDA system (EMBO J., 2001). Using this and other experimental systems, I revealed functional structure and structure-function relationship of the sliding clamp and DnaA protein in RIDA reaction. Furthermore, I search for factors re-activating ADP-DnaA protein, and found a specific DNA sequence including DnaA-binding motif as an activator. As such, this research program was carried out with important progress in analyses of DnaA regulation and the replication cycle control.