DnaA protein functions by binding to asymmetric 9mer DNA sites, the DnaA boxes. ATP-DnaA and ADPDnaA bind to 9mer DnaA boxes with equal affinity, but only ATP-DnaA protein binds in addition to an as yet unknown 6mer site, the ATP-DnaA box AGATCT, or a close match to it. ATP-DnaA protein binding to ATP-DnaA boxes is restricted to sites located in close proximity to DnaA boxes, suggesting that proteinprotein interaction is required for its stabilization. We show that ATP-DnaA represses dnaA transcription much more efficiently than ADP-DnaA. DnaA is thus a regulatory molecule that, depending on the adenosine nucleotide bound, can bind to different sequences and thereby fulfill distinct functions.

SummaryThe initiation of chromosome replication in Escherichia coli requires the recruitment of the replicative helicase DnaB from the DnaBC complex to the unwound region within the replication origin oriC, supported by the oriC-bound initiator protein DnaA. We defined physical contacts between DnaA and DnaB that involve residues 24±86 and 130±148 of DnaA and residues 154±210 and 1±156 of DnaB respectively. We propose that contacts between DnaA and DnaB occur via two interaction sites on each of the proteins. Interaction domain 24±86 of DnaA overlaps with its N-terminal homo-oligomerization domain (residues 1±86). Interaction domain 154± 210 of DnaB overlaps or is contiguous with the domains known to interact with plasmid initiator proteins. Loading of the DnaBC helicase in vivo can only be performed by DnaA derivatives containing (in addition to residues 24±86 and the DNA-binding domain 4) a structurally intact domain 3. Nucleotide binding by domain 3 is, however, not required. The parts of DnaA required for replication of pSC101 were clearly different from those used for helicase loading. Domains 1 and 4 of DnaA, but not domain 3, were found to be involved in the maintenance of plasmid pSC101.

Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo‐oligomerization of DnaA via its N‐terminus (amino acid residues 1–86) is also essential for initiation. Results from solid‐phase protein‐binding assays indicate that residues 1–86 (or 1–77) of DnaA are necessary and sufficient for self interaction. Using a ‘one‐hybrid‐system’ we found that the DnaA N‐terminus can functionally replace the dimerization domain of coliphage lambda cI repressor: a λcI‐DnaA chimeric protein inhibits λ plasmid replication as efficiently as λcI repressor. DnaA derivatives with deletions in the N‐terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N‐terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N‐termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross‐talk with the nucleotide‐binding domain III or the DNA‐binding domain IV. We propose that E. coli DnaA is composed of largely independent domains — or modules — each contributing a partial, though essential, function to the proper functioning of the ‘holoprotein’.

Bacteriophages (prokaryotic viruses) are favourite model systems to study DNA replication in prokaryotes, and provide examples for every theoretically possible replication mechanism. In addition, the elucidation of the intricate interplay of phage-encoded replication factors with 'host' factors has always advanced the understanding of DNA replication in general. Here we review bacteriophage replication based on the long-standing observation that in most known phage genomes the replication genes are arranged as modules. This allows us to discuss established model systems--f1/fd, phiX174, P2, P4, lambda, SPP1, N15, phi29, T7 and T4--along with those numerous phages that have been sequenced but not studied experimentally. The review of bacteriophage replication mechanisms and modules is accompanied by a compendium of replication origins and replication/recombination proteins (available as supplementary material online).

The replication-initiator protein DnaA is ubiquitous in the eubacterial world. It binds to an asymmetric 9 bp consensus DNA sequence, the DnaA box. Besides its primary function as an initiator, it acts as a transcription factor that represses or activates several genes, or terminates transcription, depending on the location and arrangement of DnaA boxes.