Initiator titration as a mechanism for regulation of initiation of DNA replication

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Replication of the Escherichia coli chromosome is initiated at a unique site, oriC.Concturrent initiations occur at all oriC sites present in a cell once and only once per cell cycle. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which is a 1-kb segment located at 94.7 min on the genetic map and titrates exceptionally large amounts of the bacterial initiator protein, DnaA.A strain lacking datA grew normally but exhibited the asynchronous initiation phenotype due to extra initiation events.In the present study, seven other DnaA-binding sites were examined for their possible involvement in the control of replication initiation. Disruption of the seven sites did not affect the timing of initiation of replication, even when all of them were disrupted simultaneously. Thus, datA seems to be a unique chromosomal element that appears to adjust a balance between free and bound DnaA for the single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevent reinitiations when oriC is desequestered.Mutation either in the second or in the third DnaA box (a 9-bp DnaA-binding sequence) in datA were enough to induce the mutant phenotype. Other three DnaA boxes in datA seemed to have no effect on the datA function. The second and the third DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.Regulatory inactivation of DnaA (RIDA) is another mechanism known to prevent untimely extra initiations. We found that RIDA operates independent from DnaA titration to datA.This suggests that these two mechanisms may play complementary roles during the cell cycle to ensure the scheduled initiation.