Walter Messer

Mechanism of origin unwinding: sequential binding of DnaA to double‐ and single‐stranded DNA

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.

ATP– and ADP–DnaA protein, a molecular switch in gene regulation

DnaA protein functions by binding to asymmetric 9mer DNA sites, the DnaA boxes. ATP–DnaA and ADP–DnaA 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 protein–protein 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.

DnaA initiator—also a transcription factor

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.

The DNA binding domain of the initiator protein DnaA.

The 94 C‐terminal amino acids of the initiator protein DnaA of Escherichia coli are required and sufficient for specific binding to the cognate DNA binding site. The binding domain contains two potential amphipathic alpha‐helices and a third alpha‐helix. It represents a new DNA binding motif so far not found in other DNA binding proteins. Temperature‐sensitive mutations in the binding motif, dnaA204, dnaA205 and dnaA211, abolish DNA binding. In the solid‐phase DNA binding assay, applicable to other DNA binding proteins, fusions of domains of DnaA protein to beta‐galactosidase are reacted with biotinylated anti‐beta‐galactosidase antibody. These are coupled to streptavidin‐coated magnetic beads. The DNA binding domain is able to selectively remove the DNA target (oriC) from the liquid phase. Alternatively, the DNA binding domain is fused to a peptide containing a target sequence which is naturally biotinylated in vivo in E.coli. This fusion protein can be coupled directly to streptavidin‐coated magnetic beads. Homologies between DnaA protein and transcription factors of the NtrC family are discussed.

New cloning vectors for integration into the λ attachment site attB of the Escherichia coli chromosome

Abstract A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage λ attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the λ attachment site attP and (ii) a helper plasmid, bearing the λ int gene, transcribed from the λ P R promoter under the control of the temperature-sensitive repressor c I 857 . The DNA fragment of interest is cloned into the multicloning site of one of the attP -harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 °C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 °C. These vectors have been successfully used for integration of several promoter- lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.

The interaction domains of the DnaA and DnaB replication proteins of Escherichia coli.

The 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.

The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA

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’.

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

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.

LOCALIZED DNA MELTING AND STRUCTURAL PERTUBATIONS IN THE ORIGIN OF REPLICATION, ORIC, OF ESCHERICHIA COLI IN VITRO AND IN VIVO

The leftmost region of the Escherichia coli origin of DNA replication (oriC) contains three tandemly repeated AT‐rich 13mers which have been shown to become single‐stranded during the early stages of initiation in vitro. Melting is induced by the ATP form of DnaA, the initiator protein of DNA replication. KMnO4 was used to probe for single‐stranded regions and altered DNA conformation during the initiation of DNA replication at oriC in vitro and in vivo. Unpairing in the AT‐rich 13mer region is thermodynamically stable even in the absence of DnaA protein, but only when divalent cations are omitted from the reaction. In the presence of Mg2+, oriC melting is strictly DnaA dependent. The sensitive region is distinct from that detected in the absence of DnaA as it is located further to the left within the minimal origin. In addition, the DNA is severely distorted between the three 13mers and the IHF binding site in oriC. A change of conformation can also be observed during the initiation of DNA replication in vivo. This is the first in vivo evidence for a structural change at the 13mers during initiation complex formation.

Regulation of transcription of the chromosomaldnaA gene ofEscherichia coli

SummaryBy comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomaldnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin. Inactivation of the protein indnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression ofdnaA transcription. BothdnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold. Increasing the number oforiC sequences (number of DnaA binding sites) in the cell by introducingoriC plasmids leads to a derepression of transcription. Autoregulation and binding tooriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation atoriC. The efficiency of transcription of thednaA2 promoter is reduced in the absence ofdam methylation, which is involved in the regulation oforiC replication.