The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli

Abstract

Summary Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation. Deletion of the datA locus results in extra initiations and altered temporal control of replication. There are many other sites on the E. coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated. In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation. Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication. Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle. Mutation either in the second or the third DnaA box (a 9 basepair DnaA‐binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA‐deleted strain. These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.

DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

“…We observed that datA efficiently hydrolyzed ATP bound to DnaA in a manner dependent on both IHF and Arg285 of the DnaA Argfinger, suggesting that specific DnaA multimer formation is important for DDAH. Deletion of either datA or ihf increased cellular ATP-DnaA levels in a RIDA-independent manner, consistent with the fact that untimely initiations occur in datA and IHF mutant cells (23)(24)(25)27). Because IHF binding to datA in cells was detected specifically at the postinitiation stage, datA function is likely to be temporally regulated.…”

“…1A), is crucial for repressing untimely initiation events (22)(23)(24)(25). datA-deleted cells, like seqA-deleted cells, perform untimely initiations at a level that does not inhibit cell growth (23).…”

“…DnaA binding to datA is thought to reduce the number of DnaA molecules accessible to oriC, thereby inhibiting untimely initiations (22)(23)(24)(25). Originally, datA was speculated to bind ∼370 DnaA molecules (22); however, this figure is deduced from indirect measurements not yet supported by direct evidence.…”

“…The original characterization of datA suggested that the region containing DnaA boxes 1-5 can bind the maximum number of DnaA molecules in vivo (22). Later studies found that only DnaA boxes 2-3 and IBS are crucial for repressing untimely initiations (24,25). To identify the specific sequence element prerequisites of datA for DDAH, we first performed deletion analysis using a reconstituted DDAH assay ( Fig.…”

Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis

“…A second level of regulation is mediated by the sequestration of the oriC region by the SeqA protein, which binds with a high affinity to hemimethylated GATC sequences within oriC, thus temporarily restraining reintitiation (6)(7)(8). The third level of initiation control involves the titration of the DnaA protein onto DnaA boxes at the datA locus (9,10). Indeed, datA acts to promote the correct timing of initiation relative to the cell mass (11).…”