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DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in livingEscherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC ) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of theoriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA ) from theoriC region. However, involvement ofdatA in the initiation control was suggested from the observation that, inΔdatA cells, DnaA-EYFP maximally colocalized with theoriC region earlier in the cell cycle than it did in wild-type cells andoriC concentration was increased.

Replication Initiator DnaA ofEscherichia coliChanges Its Assembly Form on the Replication Origin during the Cell Cycle