Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli

The bacterial DnaA protein binds to the chromosomal origin of replication t o trigger a series of initiation reactions, which leads to the loading of D NA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bou nd inactivated form. This negative regulation of DnaA, which occurs through interaction with the beta -subunit sliding clamp configuration of the poly merase, functions in the temporal blocking of re-initiation. Here we show t hat the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal rep lication and for cell viability. Hda belongs to the chaperone-like ATPase f amily, AAA(+), as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rul e of replication depends on the timely interaction of AAA(+) proteins that comprise the apparatus regulating the activity of the initiator of replicat ion.