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’.

Using an electron microscopic method for visualizing interactions of the Escherichia coli DnaA protein with weak DnaA-binding DNA sequences, we found that DnaA binds effectively to two separated weak DnaA box regions located on the same DNA fragment. As expected, no DnaA-DNA interactions were detected when both DnaA box regions were mutagenized to the sequence totally incapable of binding DnaA. However, when only one of these two regions was mutagenized, the lack of interactions between DnaA and DNA was observed not only at the scrambled DnaA box but also at the second weak DnaA box region. These results indicate that for the efficient binding of DnaA to a weak DnaA box region, the presence of at least two such DNA sequences is necessary. Our finding also suggests that binding of DnaA protein to weak DnaA box sequences may be cooperative. In addition, we found that DnaA-mediated transcription termination in vivo requires two DnaA boxes, one of them is a weak one. It seems, therefore, that some mechanisms of regulation of transcription and DNA replication by DnaA, that involve interactions of DnaA with weak DnaA boxes, may be more complicated than initially proposed.

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1–86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87–134, constitutes a flexible loop. Domain 3, aa 135–373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band‐shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9‐mer repeats) in oriC, and by a competition band‐shift assay. DnaA box R4 (oriC positions 260–268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild‐type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low‐affinity site a novel candidate for a regulatory DnaA box.