DnaA Boxes Research Articles

Strand-specific loading of DnaB helicase by DnaA to a substrate mimicking unwound oriC.

We analysed the enzymatic activity (strand dis-placement) of the Escherichia coli DnaB helicase on a mirror-image pair of oligonucleotide-based substrates mimicking the unwound replication origin oriC. Loading of the helicase complex occurred exclusively to the single-stranded 'lower strand' part of the substrates. Full helicase activity required DnaA bound to the double-stranded part of the substrates (oriC DnaA box R1) and to their single-stranded 'upper strand' part. We assume that in vivo DnaA also loads the first of two helicase complexes - required for the assembly of two replication forks - to the lower strand of oriC during initiation of bidirectional chromosome replication in E. coli.

Escherichia coli DnaA Protein Loads a Single DnaB Helicase at a DnaA Box Hairpin

The molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB. At oriC, two and only two helicase molecules are loaded, one for each replication fork. DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase. Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind. In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused. We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure.

Two dnaB genes are associated with the origin of replication of pQBR55, an exogenously isolated plasmid from the rhizosphere of sugar beet

Plasmid pQBR55 (∼149 kb) represents one of five (Groups I–V) genetically distinct transfer proficient, mercury resistance plasmid groups that have been observed in the phytosphere pseudomonad community at a single geographic location in Oxford, UK. A 4.9-kb HindIII fragment was cloned from pQBR55 (a Group III plasmid) that facilitates autonomous replication of a narrow host range cloning vector, pKIL29, in the non-permissive host Pseudomonas putida UWC1. Sequencing revealed that the fragment contains a unique tandem array of dnaB genes (one partial and one complete), a homologue of the traA gene of plasmid RP4 and several potential open reading frames with little homology to any known sequences. The fragment also contains: a short region of direct and indirect repeats, two sequences that resemble the consensus Escherichia coli DnaA box motif, an A+T rich region and evidence of a GC-skew inversion. All features associated with origins of replication. Two specific oligonucleotide primer pairs, one targeted at the tandem dnaB genes and the other at the dnaB–traA arrangement were used for PCR analysis of other plasmids isolated from the same field site. PCR generated amplification products were only amplified from Group III plasmids (defined by RFLP typing) but not from plasmids belonging to Group I, II, IV or V. The nucleotide sequences of the amplified fragments were identical to those of pQBR55, even though the other Group III plasmids had distinct RFLP patterns. The results of a more general PCR screen, using primers to amplify a 389-bp fragment of all described pseudomonad dnaB genes, suggests that closely related dnaB sequences are only associated with Group III plasmids.

Two dnaB genes are associated with the origin of replication of pQBR55, an exogenously isolated plasmid from the rhizosphere of sugar beet

Plasmid pQBR55 ( approximately 149 kb) represents one of five (Groups I-V) genetically distinct transfer proficient, mercury resistance plasmid groups that have been observed in the phytosphere pseudomonad community at a single geographic location in Oxford, UK. A 4.9-kb HindIII fragment was cloned from pQBR55 (a Group III plasmid) that facilitates autonomous replication of a narrow host range cloning vector, pKIL29, in the non-permissive host Pseudomonas putida UWC1. Sequencing revealed that the fragment contains a unique tandem array of dnaB genes (one partial and one complete), a homologue of the traA gene of plasmid RP4 and several potential open reading frames with little homology to any known sequences. The fragment also contains: a short region of direct and indirect repeats, two sequences that resemble the consensus Escherichia coli DnaA box motif, an A+T rich region and evidence of a GC-skew inversion. All features associated with origins of replication. Two specific oligonucleotide primer pairs, one targeted at the tandem dnaB genes and the other at the dnaB-traA arrangement were used for PCR analysis of other plasmids isolated from the same field site. PCR generated amplification products were only amplified from Group III plasmids (defined by RFLP typing) but not from plasmids belonging to Group I, II, IV or V. The nucleotide sequences of the amplified fragments were identical to those of pQBR55, even though the other Group III plasmids had distinct RFLP patterns. The results of a more general PCR screen, using primers to amplify a 389-bp fragment of all described pseudomonad dnaB genes, suggests that closely related dnaB sequences are only associated with Group III plasmids.

Mutations in the CCGTTCACA DnaA box of Mycobacterium tuberculosis oriC that abolish replication of oriC plasmids are tolerated on the chromosome.

The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements. To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains. Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M. tuberculosis. Furthermore, by using a surface plasmon resonance technique we showed that purified M. tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M. tuberculosis H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that M. tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M. tuberculosis oriC replication are different for chromosomes and plasmids.

OriC Region and Replication Termination Site, dif , of the Xanthomonas campestris pv. campestris 17 Chromosome

A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X. campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage phiLf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication.

The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli.

Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions. 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, to prevent overinitiation. Deletion of the datA locus results in extra initiations and altered temporal control of replication. There are many other sites on the E. coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated. In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation. Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication. Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle. Mutation either in the second or the third DnaA box (a 9 basepair DnaA-binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA-deleted strain. These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.

Stoichiometry of DnaA and DnaB Protein in Initiation at the Escherichia coli Chromosomal Origin

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.

A hybrid bacterial replication origin.

We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC. The AT-rich region could be unwound by E. coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble. The results show that species specificity, i.e. which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.

DnaA Box Sequences as the Site for Helicase Delivery during Plasmid RK2 Replication Initiation in Escherichia coli

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.

DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number

The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.

Identification of a putative chromosomal replication origin from Helicobacter pylori and its interaction with the initiator protein DnaA.

The key elements of the initiation of Helicobacter pylori chromosome replication, DnaA protein and putative oriC region, have been characterized. The gene arrangement in the H.pylori dnaA region differs from that found in many other eubacterial dnaA regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB). Helicobacter pylori dnaA is flanked by two open reading frames with unknown function, while dnaN-gyrB and rnpA-rmpH loci are separated from the dnaA gene by 600 and 90 kb, respectively. We show that the dnaA gene encoding initiator protein DnaA is expressed in H.pylori cells. The H.pylori DnaA protein, like other DnaA proteins, can be divided into four domains. Here we demonstrate that the C-terminal domain of H.pylori DnaA protein is responsible for DNA binding. Using in silico and in vitro studies, the putative oriC region containing five DnaA boxes has been located upstream of the dnaA gene. DNase I and gel retardation analyses show that the C-terminal domain of H.pylori DnaA protein specifically binds each of five DnaA boxes.

Characterization and Sequence Analysis of the Replicator Region of the Novel Plasmid pALC1 from Paracoccus alcaliphilus

The replicator region of a low-copy-number plasmid, pALC1, of Paracoccus alcaliphilus JCM 7364 was cloned in a form of the minireplicon pALC100 (3.6 kb). The host range of the minireplicon embraces several species of genus Paracoccus, as well as Agrobacterium tumefaciens, Rhizobium leguminosarum, and Rhodobacter sphaeroides (all belonging to α-Proteobacteria), but not Escherichia coli. The complete nucleotide sequence of the replicator region (2276 bp) revealed the presence of one complete open reading frame coding for the 28.4-kDa protein (RepA) with similarity to replication proteins of plasmid pSW500 of Erwinia stewartii and pVS1 of Pseudomonas fluorescens. The iteron-like region was identified upstream of the repA gene and consisted of two clusters of repeated sequences (17 bp long) separated by a putative DnaA box. Analysis of the predicted amino acid sequence of two adjacent incomplete ORFs suggests the localization of repA between genes involved in conjugation (traG) and partitioning (parA) within the pALC1 genome.

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome.

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.

Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene.

Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle. Like most eubacteria, C. crescentus has a DnaA homologue that is presumed to initiate chromosome replication. However, the C. crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes. Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator. We therefore created a conditional dnaA C. crescentus strain. Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period. Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage. Cell division also stopped, but not swarmer to stalked cell differentiation. All cells became stalked cells that grew as long filaments. Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped. However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors. Interestingly, transcription from Cori Ps continued unaltered. Therefore, Ps transcription is not sufficient for chromosome replication. Approximately 6-8 h after blocked dnaA expression, cells lost viability exponentially. Coincidentally, beta-galactosidase was induced from one transcription reporter, suggesting an altered physiology. We conclude that C. crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.

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'-TT(A/T)TNCACA). 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 approximately 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.

Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces

Using a combined PCR-gel retardation assay, the preferred recognition sequence of the Streptomyces initiator protein DnaA was determined. The protein showed a preference toward DNA containing two Escherichia coli-like DnaA boxes in a head-to-head arrangement (consensus sequence TTATCCACA, whereas the consensus sequence of the DnaA boxes found in the Streptomyces oriC region is TTGTCCACA). In quantitative band shift experiments, the kinetics of the Streptomyces DnaA-DnaA box interaction was characterized. The DnaA protein can form dimers while binding to a single DnaA box; dimer formation is mediated by the domain III of the protein, and the dissociation constant of this process was between 35 and 115 nm. Streptomyces initiator protein DnaA interacts in a cooperative manner with DNA containing multiple binding sites. For the cooperativity effect, which seems to be independent of the distance separating the DnaA boxes, domain I (or I and II) is responsible. The cooperativity constant is moderate and is in the range of 20-110.

Anomalous DnaA protein binding to the regulatory region of the Escherichia coli aldA gene.

A binding site for DnaA protein was identified in the regulatory region of the aldA gene of Escherichia coli. Specific binding was demonstrated by in vitro assays including filter binding as well as mobility shift in a polyacrylamide gel of the DnaA-DNA complex. In cells growing in minimal medium containing glucose, expression of ss-galactosidase activity from an aldA-lacZ fusion gene was suppressed by oversupply of DnaA protein and was enhanced by reducing the free DnaA level. These results suggest that DnaA protein negatively regulates expression of the aldA gene under these conditions. Despite fairly strong binding, the bound DNA fragment had no consensus 9 bp DnaA binding sequence (DnaA box), and anomalous binding to an AT-rich sequence located close to the transcription start site was revealed by a footprinting experiment.

Identification and characterization of the dnaA upstream region of Thermus thermophilus

The gene order in the dnaA region of Thermus thermophilus was determined. Previously, we showed that the putative oriC of T. thermophilus is located in the dnaA- dnaN intergenic region. In the 4 kb region upstream of the dnaA gene four ORFs were found, all orientated in the same direction which is opposite to that of dnaA. The ORFs were identified as T. thermophilus homologs of gidA, gidB, soj and spo0J of Bacillus subtilis. The gene order spo0J-soj-gidB-gidA-dnaA-dnaN resembles that of B. subtilis, Pseudomonas putida, Coxiella burnetii, Streptomyces coelicolor, Mycobacterium leprae, and Mycobacterium tuberculosis. We identified the transcriptional start point of the dnaA gene. The -10 region shows significant homology to the Escherichia coli -10 consensus sequence. The putative -35 region shows homology neither to the E. coli -35 consensus sequence nor to known -35 sequences of T. thermophilus. There are no DnaA boxes in the promoter region, and consequently dnaA transcription is not repressed by DnaA protein in vitro, i.e. the dnaA gene of T. thermophilus is not autoregulated.

Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.

Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo. We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.