Soon after the initiation of DNA replication, the ATPase activity of the AAA+ domain of DnaA is stimulated by the essential Hda protein, resulting in the hydrolysis of the ATP bound to DnaA and thus in the regulatory inactivation of DnaA . Hda supposedly needs to interact with the b-clamp of the DNA polymerase III loaded onto DNA to promote the inactivation of DnaA . Although there exists several other mechanisms that regulate the timing of the initiation of DNA replication in E. coli , the RIDA system seems to be one of the most important . Consistent with this model, it was shown that ATPase defective mutants of DnaA, and hda-deficient cells, display overinitiation phenotypes in E. coli . The use of replisomeassociated proteins that act as negative regulators of initiation appears quite conserved in bacteria, as indicated by the discoveries of the YabA protein in Bacillus subtilis or the HdaA protein in Caulobacter crescentus . The C. crescentus bacterium undergoes a unique developmental cycle, dividing asymmetrically at each cell cycle, producing two different daughter cells: the stalked cell in S phase, and the swarmer cell in G1 phase until it differentiates into a stalked cell . The G1-to-S transition is controlled by the CtrA protein, an important purchase SB431542 regulator of the initiation of chromosome replication in alphaproteobacteria. This response regulator binds to several sites in the C. crescentus origin of replication to inhibit the initiation of DNA replication, probably by competing with DnaA binding . It also binds to many promoters to regulate their activities . Levels of the active phosphorylated form of CtrA are tightly controlled by the regulated synthesis, phosphorylation and degradation of the CtrA protein, so that it only GW-572016 accumulates in swarmer and pre-divisional cells . A gradient of active CtrA establishes the asymmetry of the initiation of DNA replication between the two future daughter cells before cell division . Active CtrA is however undetectable for about one third of the cell cycle during the stalked cell stage . During the time when no CtrA is detectable, other factors restrict the initiation of chromosomal replication to only once per cell cycle. We previously identified HdaA as one of these factors, showing that the HdaA protein is necessary to ensure that chromosomal replication does not restart a second time during the same cell cycle , indicating that HdaA may stimulate the inactivation of DnaA once DNA replication has initiated, by a mechanism similar to the RIDA system in E. coli. To further address this possibility, we describe here the creation of a mutant DnaA protein in C. crescentus, with an R357A substitution in its AAA+ domain. We found that the expression of this protein caused severe over-initiation of chromosomal replication and blocked cell division in C. crescentus, unlike the expression of the wild-type DnaA, indicating that DnaA is hyper-active as an initiator of DNA replication.