Mechanism for the TtDnaA-Tt-oriC cooperative interaction at high temperature and duplex opening at an unusual AT-rich region in Thermoanaerobacter tengcongensis

Thermoanaerobacter tengcongensis is an anaerobic low-GC thermophilic bacterium. To further elucidate the replication initiation of chromosomal DNA at high temperature, the interaction between the replication initiator (TtDnaA) and the putative origin (Tt-oriC) in this thermophile was investigated. We found that efficient binding of TtDnaA to Tt-oriC at high temperature requires (i) at least two neighboring DnaA boxes, (ii) the specific feature of the TtDnaA Domain IV and (iii) the self-oligomerization of TtDnaA. Replacement of the TtDnaA Domain IV by the counterpart of Escherichia coli DnaA or disruption of its oligomerization by amino acid mutations (W9A/L20S) abolished the oriC-binding activity of TtDnaA at 60°C, but not at 37°C. Moreover, ATP-TtDnaA, but not ADP-TtDnaA or the oligomerization-deficient mutants was able to unwind the Tt-oriC duplex. The minimal oriC required for this duplex opening in vitro was demonstrated to consist of DnaA boxes 1–8 and an unusual AT-rich region. Interestingly, although no typical ATP-DnaA box was found in this AT-rich region, it was exclusively bound by ATP-TtDnaA and acted as the duplex-opening and replication-initiation site. Taken together, we propose that oligomerization of ATP-DnaA and simultaneously binding of several DnaA boxes and/or AT-rich region may be generally required in replication initiation at high temperature.     

Mutations in the DnaA binding sites of the replication origin of Escherichia coli.

Summary Mutations (base changes) were introduced into the four DnaA binding sites (DnaA boxes) of theEscherichia coli replication origin,oriC. Mutations in a single DnaA box did not impair the ability of these origins to replicate in vivo and in vitro. A combination of mutations in two DnaA boxes, R1 and R4, resulted in slower growth of theoriC plasmid-bearing host cells. DnaA protein interaction with mutant and wild-type DnaA boxes was analyzed by DNase I footprinting. Binding of DnaA protein to a mutated DnaA box R1 was not affected by a mutation in DnaA box R4 and vice versa. Mutations in DnaA boxes R1 and R4 did not modify the ability of the DnaA protein to bind to other DnaA boxes inoriC.     

The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50^° bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaC complex, much as has been documented for oriC- dependent replication.     

DiaA/HobA and DnaA: a pair of proteins co-evolved to cooperate during bacterial orisome assembly

Replication of the bacterial chromosome is initiated by binding the DnaA protein to oriC. Various factors control the ability of DnaA to bind and unwind DNA. Among them, Escherichia coli DiaA and Helicobacter pylori HobA have been characterized recently. They were found to interact with domain I of DnaA and stimulate DnaA binding to oriC. We examined HobA and DiaA functional homology and showed that, despite a high degree of structural similarity, they are not interchangeable because they are unable to interact with heterologous DnaA proteins. We revealed particular structural differences impeding formation of heterologous complexes and, consistently, we restored DiaA-enhanced oriC binding by the hybrid Ec^I-Hp^II-IVDnaA protein; i.e. H. pylori DnaA in which domain I was exchanged with that of E. coli. This proved that DiaA and HobA are functional homologs and upon binding to DnaA they exert a similar effect on orisome formation. Interestingly, we showed for the first time that the dynamics of DiaA- and HobA-stimulated orisome assembly are different. HobA enhances and accelerates HpDnaA binding to oriC, whereas DiaA increases but decelerates EcDnaA binding with oriC. We postulate that the different dynamics of orisome formation reflect the distinct strategies adopted by E. coli and H. pylori to regulate the frequency of the replication of their chromosomes. DiaA/HobA homolog have been identified in many proteobacteria and therefore might constitute a common, though species-specific, factor modulating bacterial orisome assembly.     

Structural and thermodynamic signatures of DNA recognition by Mycobacterium tuberculosis DnaA

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 Å resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 Å). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.     

DnaA structure, function, and dynamics in the initiation at the chromosomal origin

Escherichia coli DnaA is the initiator of chromosomal replication. Multiple ATP-DnaA molecules assemble at the oriC replication origin in a highly regulated manner, and the resultant initiation complexes promote local duplex unwinding within oriC, resulting in open complexes. DnaB helicase is loaded onto the unwound single-stranded region within oriC via interaction with the DnaA multimers. The tertiary structure of the functional domains of DnaA has been determined and several crucial residues in the initiation process, as well as their unique functions, have been identified. These include specific DNA binding, inter-DnaA interaction, specific and regulatory interactions with ATP and with the unwound single-stranded oriC DNA, and functional interaction with DnaB helicase. An overall structure of the initiation complex is also proposed. These are important for deepening our understanding of the molecular mechanisms that underlie DnaA assembly, oriC duplex unwinding, regulation of the initiation reaction, and DnaB helicase loading. In this review, we summarize recent progress on the molecular mechanisms of the functions of DnaA on oriC. In addition, some members of the AAA+ protein family related to the initiation of replication and its regulation (e.g., DnaA) are briefly discussed.     

Helicobacter pylori oriC��the first bipartite origin of chromosome replication in Gram-negative bacteria

Binding of the DnaA protein to oriC leads to DNA melting within the DNA unwinding element (DUE) and initiates replication of the bacterial chromosome. Helicobacter pylori oriC was previously identified as a region localized upstream of dnaA and containing a cluster of DnaA boxes bound by DnaA protein with a high affinity. However, no unwinding within the oriC sequence has been detected. Comprehensive in silico analysis presented in this work allowed us to identify an additional region (oriC2), separated from the original one (oriC1) by the dnaA gene. DnaA specifically binds both regions, but DnaA-dependent DNA unwinding occurs only within oriC2. Surprisingly, oriC2 is bound exclusively as supercoiled DNA, which directly shows the importance of the DNA topology in DnaA-oriC interactions, similarly as previously presented only for initiator-origin interactions in Archaea and some Eukaryota. We conclude that H. pylori oriC exhibits bipartite structure, being the first such origin discovered in a Gram-negative bacterium. The H. pylori mode of initiator-oriC interactions, with the loop formation between the subcomplexes of the discontinuous origin, resembles those discovered in Bacillus subtilis chromosome and in many plasmids, which might suggest a similar way of controlling initiation of replication.     

Differentiation of the DnaA-oriC Subcomplex for DNA Unwinding in a Replication Initiation Complex

In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.     

The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

ATP-DnaA binds to multiple DnaA boxes in the Escherichia coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg-285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that had alternative DNA binding sequence specificity and chimeric oriC molecules bearing the alternative DnaA binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg-285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg-285 fingers face inward to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases.     

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.     

The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli

 Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50^° bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaC complex, much as has been documented for oriC- dependent replication. Received: 15 May 1996/Accepted: 11 July 1996     

Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in ΔdatA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.Initiation of DNA replication is highly regulated to coordinate with cell proliferation. It begins with a series of events in which the replication machinery is assembled at the replication origin of the chromosomal DNA (15, 26, 28, 38). Central to this process are the initiator proteins that bind to the origin of replication and eventually lead to the unwinding of the origin and to helicase loading on the unwound region. Previous biochemical studies and recent structural studies of the bacterial initiator protein DnaA have proposed the molecular mechanism of the action of ATP-DnaA in forming a large oligomeric complex to remodel the unique origin, oriC, and trigger duplex melting (12, 26). However, it is still not clear how the timing of initiation is controlled so that it takes place at a fixed time in the cell cycle. It has been reported that a basal level of DnaA molecules is bound by high-affinity DnaA binding sites (DnaA boxes R1, R2, and R4) at oriC throughout the cell cycle (9, 37). It is also suggested that noncanonical ATP-DnaA binding sites within oriC are occupied at elevated levels of the initiator molecules prior to the initiation event (18, 25). Thus, regulation of the activity and availability of DnaA is an important factor for the initiation control.At least three schemes are known to prevent untimely initiations in Escherichia coli. First, oriC is subject to sequestration, a process that prevents reinitiation, possibly by blocking ATP-DnaA from binding to newly replicated oriC (8, 24). E. coli oriC contains 11 GATC sites that are normally methylated on both strands by Dam methyltransferase. Immediately after passage of the replication fork, GATC sites are in a hemimethylated state, with the newly synthesized strands remaining unmethylated. SeqA binds specifically to such sites and, at oriC, protects these regions from reinitiation for about one-third of the cell cycle (6, 39). Second, in a process termed regulatory inactivation of DNA (RIDA), ATP-DnaA molecules are converted to an inactive ADP-bound form after initiation by the combined action of a β subunit of DNA polymerase III holoenzyme and Hda (16, 17). Newly synthesized DnaA molecules are able to bind ATP for the next initiation event, since its cellular concentration is much higher than that of ADP. ATP-DnaA is also regenerated from the inactive ADP-DnaA later in the cell cycle (21). Finally, the chromosomal segment datA serves to reduce the level of free DnaA protein by titrating a large number of DnaA molecules after replication of the site close to oriC (20).Cytological studies would be very useful for developing our understanding of the regulation mechanisms associated with the initiation step. In the present study, we tagged E. coli DnaA with a fluorescent protein in order to monitor its behavior in live cells. Microscopic observation revealed that DnaA is distributed all over the nucleoid. Remarkably, the majority of cells bore distinct foci that emitted brighter fluorescence against a weak fluorescent background on the nucleoid. We analyzed the behavior of these foci during the cell cycle with respect to oriC and datA.     

Assembly of Helicobacter pylori Initiation Complex Is Determined by Sequence-Specific and Topology-Sensitive DnaA–oriC Interactions

In bacteria, chromosome replication is initiated by binding of the DnaA initiator protein to DnaA boxes located in the origin of chromosomal replication (oriC). This leads to DNA helix opening within the DNA-unwinding element. Helicobacter pylori oriC, the first bipartite origin identified in Gram-negative bacteria, contains two subregions, oriC1 and oriC2, flanking the dnaA gene. The DNA-unwinding element region is localized in the oriC2 subregion downstream of dnaA. Surprisingly, oriC2–DnaA interactions were shown to depend on DNA topology, which is unusual in bacteria but is similar to initiator–origin interactions observed in higher organisms. In this work, we identified three DnaA boxes in the oriC2 subregion, two of which were bound only as supercoiled DNA. We found that all three DnaA boxes play important roles in orisome assembly and subsequent DNA unwinding, but different functions can be assigned to individual boxes. This suggests that the H. pylori oriC may be functionally divided, similar to what was described recently for Escherichia coli oriC. On the basis of these results, we propose a model of initiation complex formation in H. pylori.     

Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens,Mycoplasma bovis and Mycoplasma agalactiae

Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.     

The complex for replication initiation of<Emphasis Type="Italic">Escherichia coli</Emphasis>

We probed the complex betweenoriC and DnaA protein using two types of mutants inoriC. Base changes in the DnaA binding sites, DnaA boxes, had little effect on origin function. Mutations which change the distance between DnaA boxes R3 and R4, on the other hand, inactivatedoriC unless the mutation deleted or inserted one complete helical turn. Origins with other 10 base pair insertions in the interval between DnaA boxes R2 and R3 were functional, but not insertions in the R1–R2 interval. FIS protein binds to a bipartite site inoriC between DnaA boxes R2 and R3. A model for theoriC/DnaA complex based on these results suggests an array of DnaA monomers with a 34 Å spacing upon whichoriC is arranged.     

Structural basis of replication origin recognition by the DnaA protein

Escherichia coli DnaA binds to 9 bp sequences (DnaA boxes) in the replication origin, oriC, to form a complex initiating chromosomal DNA replication. In the present study, we determined the crystal structure of its DNA-binding domain (domain IV) complexed with a DnaA box at 2.1 Å resolution. DnaA domain IV contains a helix–turn–helix motif for DNA binding. One helix and a loop of the helix– turn–helix motif are inserted into the major groove and 5 bp (3′ two-thirds of the DnaA box sequence) are recognized through base-specific hydrogen bonds and van der Waals contacts with the C5-methyl groups of thymines. In the minor groove, Arg399, located in the loop adjacent to the motif, recognizes three more base pairs (5′ one-third of the DnaA box sequence) by base-specific hydrogen bonds. DNA bending by ~28° was also observed in the complex. These base-specific interactions explain how DnaA exhibits higher affinity for the strong DnaA boxes (R1, R2 and R4) than the weak DnaA boxes (R3 and M) in the replication origin.     

dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.     

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 Lf 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.     

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinusdnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them.     

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.