In Escherichia coli, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the datA site. The datA site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in ⌬datA cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the datA site is not required to prevent reinitiation and limit initiation of replication to once per generation. The datA site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.Chromosome replication of Escherichia coli is initiated at a single origin, oriC, by the initiator protein, DnaA (28). In rapidly growing cells, rounds of replication overlap and two, four, or even eight origins coexist and are initiated simultaneously only once per cell cycle (45). The new origins are immediately inactivated by sequestration. Sequestration relies on binding of the SeqA protein to the newly replicated, hemimethylated origins. Subsequent methylation by Dam methylase ends the sequestration (12,35,50). Sequestration is one of the processes necessary to limit initiation of replication to once and only once per cell cycle. In addition to the sequestration, a reduction of the initiation potential (i.e., sufficient amounts of active DnaA) must be achieved during the sequestration period, such that when origins are released from sequestration they cannot be reinitiated (46), and initiation cannot occur again until one generation later, when enough initiation potential again has accumulated. In this way the E. coli cell does not need to keep track of how many chromosomal origins have been initiated every generation; it is sufficient to first initiate all and then sequester all origins. This explains why E. coli can harbor large numbers of minichromosomes (oriC plasmids) without problems of incompatibility. The minichromosomal origins are initiated in synchrony with the chromosomal origins (30, 31) and sequestered in the same way.The reduction in initiation potential during sequestration can be achieved in two ways, (i) by reducing the availability of DnaA and (ii) by reducing the activity of DnaA. The availability of DnaA at oriC is affected by binding sites (DnaA boxes) distributed around the E. coli chromosome (19). One site in particular, datA, with five DnaA boxes, titrates unusually large amounts of DnaA protein in vivo (24) and is the main contributor to the DnaA ...