Equilibrium and kinetic rate constants were determined for the binding of the initiator protein DnaA of Escherichia coli to its binding site, the non-palindromic 9-bp DnaA box, using gel retardation techniques. The dissociation constant for specific binding was between 1 and 50 nM for individual DnaA boxes on 21-bp double-stranded oligonucleotides. Only DnaA boxes of the sequence TT(A/T)TNCACA resulted in specific fragment retention. Both the 9-bp consensus sequence and flanking sequences determined the binding efficiency. One DnaA monomer was found to bind to a DnaA box and to induce a bend of about 40 degrees.

The initiator protein DnaA of Escherichia coli binds to a 9mer consensus sequence, the DnaA box (5′‐TTA/TTNCACA). If complexed with ATP it adopts a new binding specificity for a 6mer consensus sequence, the ATP‐DnaA box (5′‐AGatct). Using DNase footprinting and surface plasmon resonance we show that binding to ATP‐DnaA boxes in the AT‐rich region of oriC of E.coli requires binding to the 9mer DnaA box R1. Cooperative binding of ATP‐DnaA to the AT‐rich region results in its unwinding. ATP‐DnaA subsequently binds to the single‐stranded region, thereby stabilizing it. This demonstrates an additional binding specificity of DnaA protein to single‐stranded ATP‐DnaA boxes. Binding affinities, as judged by the DnaA concentrations required for site protection in footprinting, were ∼1 nM for DnaA box R1, 400 nM for double‐stranded ATP‐DnaA boxes and 40 nM for single‐stranded ATP‐DnaA boxes, respectively. We propose that sequential recognition of high‐ and low‐affinity sites, and binding to single‐stranded origin DNA may be general properties of initiator proteins in initiation complexes.

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.

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