Role of the initiator titration mechanism in the initiation of chromosomal replication

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Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiations occur at all oriC sites present in a cell once and only once at a fixed time in the cell cycle. 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, A strain lacking datA grew normally but exhibited the asynchronous initiation phenotype due to extra initiation events. The DnaA titration mechanism to control replication initiation appears to be, intrinsic to datA, inasmuch as deletion of other DnaA-binding sites (DnaA boxes) on the chromosome does not provoke such aberrant initiations.In the present study, efforts were made to clarify the unique mechanism of DnaA titration by datA. Analyses of phenotypes of strains carrying mutations at various sites in datA revealed that, of the five DnaA boxes in datA, both the second and the third boxes are essential for the initiation control. A consensus sequence of binding of the integration host factor (IHF) is present between the two DnaA boxes. We have proven that IHF protein really binds to this site. Furthermore, the IHF binding greatly enhanced the amounts of DnaA molecules titrated to datA. Binding of IHF to its recognition site has been known to cause DNA bending of about 160 degree. This bending should bring the second and the third DnaA boxes to close proximity and enable bridging the DNA strands by DnaA-DnaA interaction bound to the two DnaA boxes, leading to the formation of a large DnaA titration pool.Destruction of the IHF-binding site in datA on the chromosome caused overinitiation phenotype similar to that observed with the datA-deleted strain, demonstrating the involvement of IHF in DnaA titration to datA in vivo.