Replication Initiation in Bacteria.

The initiation of chromosomal DNA replication starts at a replication origin, which in bacteria is a discrete locus that contains DNA sequence motifs recognized by an initiator protein whose role is to assemble the replication fork machinery at this site. In bacteria with a single chromosome, DnaA is the initiator and is highly conserved in all bacteria. As an adenine nucleotide binding protein, DnaA bound to ATP is active in the assembly of a DnaA oligomer onto these sites. Other proteins modulate DnaA oligomerization via their interaction with the N-terminal region of DnaA. Following the DnaA-dependent unwinding of an AT-rich region within the replication origin, DnaA then mediates the binding of DnaB, the replicative DNA helicase, in a complex with DnaC to form an intermediate named the prepriming complex. In the formation of this intermediate, the helicase is loaded onto the unwound region within the replication origin. As DnaC bound to DnaB inhibits its activity as a DNA helicase, DnaC must dissociate to activate DnaB. Apparently, the interaction of DnaB with primase (DnaG) and primer formation leads to the release of DnaC from DnaB, which is coordinated with or followed by translocation of DnaB to the junction of the replication fork. There, DnaB is able to coordinate its activity as a DNA helicase with the cellular replicase, DNA polymerase III holoenzyme, which uses the primers made by primase for leading strand DNA synthesis.

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.

Essential amino acids of Escherichia coli DnaC protein in an N-terminal domain interact with DnaB helicase.

Escherichia coli DnaC protein bound to ATP forms a complex with DnaB protein. To identify the domain of DnaC that interacts with DnaB, a genetic selection was used based on the lethal effect of induced dnaC expression and a model that inviability arises by the binding of DnaC to DnaB to inhibit replication fork movement. The analysis of dnaC alleles that preserved viability under elevated expression revealed an N-terminal domain of DnaC involved in binding to DnaB. Mutant proteins bearing single amino acid substitutions (R10P, L11Q, L29Q, S41P, W32G, and L44P) that reside in regions of predicted secondary structure were inert in DNA replication activity because of their inability to bind to DnaB, but they retained ATP binding activity, as indicated by UV cross-linking to [alpha-(32)P]ATP. These alleles also failed to complement a dnaC28 mutant. Other selected mutations that map to regions carrying Walker A and B boxes are expected to be defective in ATP binding, a required step in DnaB-DnaC complex formation. Lastly, we found that the sixth codon from the N terminus encodes aspartate, resolving a reported discrepancy between the predicted amino acid sequence based on DNA sequencing data and the results from N-terminal amino acid sequencing (Nakayama, N., Bond, M. W., Miyajima, A., Kobori, J., and Arai, K. (1987) J. Biol. Chem. 262, 10475-10480).

Escherichia coli DnaA protein. The N-terminal domain and loading of DnaB helicase at the E. coli chromosomal origin.

Initiation of DNA replication at the Escherichia coli chromosomal origin occurs through an ordered series of events that depends first on the binding of DnaA protein, the replication initiator, to DnaA box sequences followed by unwinding of an AT-rich region. A step that follows is the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We show that DnaA protein actively mediates the entry of DnaB at oriC. One region (amino acids 111-148) transiently binds to DnaB as determined by surface plasmon resonance. A second functional domain, possibly involving formation of a unique nucleoprotein structure, promotes the stable binding of DnaB during the initiation process and is inactivated in forming an intermediate termed the prepriming complex by removal of the N-terminal 62 residues. Based on similarities in the replication process between prokaryotes and eukaryotes, these results suggest that a similar mechanism may load the eukaryotic replicative helicase.

The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin.

The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.

Role of adenine nucleotides, molecular chaperones and chaperonins in stabilization of DnaA initiator protein of Escherichia coli.

DnaA protein of Escherichia coli is a sequence-specific DNA binding protein required for the initiation of DNA replication from the chromosomal origin, oriC, and of several E. coli plasmids. At a moderate ionic strength, purified DnaA protein has a strong tendency to aggregate; the self-aggregate form is inactive in DNA replication. Binding of ATP or ADP to DnaA protein protected it from aggregation to maintain its replication activity. AMP or cyclic AMP had no protective effect. The molecular chaperone DnaK protected DnaA protein from aggregation with or without ATP. DnaJ and GrpE were not stimulatory. Chaperonins GroEL and GroES were also able to prevent aggregation but only in the presence of ATP. The studies presented here show that for DnaA protein to be active in the initiation of DNA replication, it must be prevented from forming a self-aggregate by the binding of adenine nucleotides, and/or by the action of molecular chaperones.

Threonine 435 of Escherichia coli DnaA protein confers sequence-specific DNA binding activity.

The Escherichia coli DnaA protein, as a sequence-specific DNA binding protein, promotes the initiation of chromosomal replication by binding to four asymmetric 9-mer sequences termed DnaA boxes in oriC. Characterization of N-terminal, C-terminal, and internal in-frame deletion mutants identified residues near the C terminus of DnaA protein required for DNA binding. Furthermore, genetic and biochemical characterization of 11 missense mutations mapping within the C-terminal 89 residues indicated that they were defective in DNA binding. Detailed biochemical characterization of one mutant protein bearing a threonine to methionine substitution at position 435 (T435M) revealed that it retained only nonspecific DNA binding activity, suggesting that threonine 435 imparts specificity in binding. Finally, T435M was inactive on its own for in vitro replication of an oriC plasmid but was able to augment limiting levels of wild type DnaA protein, consistent with the proposal that not all of the DnaA monomers in the initial complex are bound specifically to oriC and that direct interaction occurs among monomers.

Novel alleles of the Escherichia coli dnaA gene.

The Escherichia coli dnaA gene is required for replication of the bacterial chromosome. To identify residues critical for its replication activity, a method to select novel mutations was developed that relied on lytic growth of lambda from an inserted pSC101 replication origin. Replication from the lambda origin was inhibited by lysogen-encoded cI repressor. Replication from the pSC101 origin that resulted in lytic growth was dependent on active DnaA protein encoded by a plasmid in a host strain lacking the chromosomal dnaA gene. With this approach, a large collection of missense, nonsense, and a few internal deletion mutations were obtained. Nucleotide sequence analysis of the missense mutations indicated that 28 of 50 were unique. Of these, one was identical to the dnaA205 allele whereas the remainder are novel. These missense mutations were clustered into three regions, suggesting three functional domains of DnaA protein required for its replication activity. Many of the missense mutations mapping to the C-terminal 61 residues were inactive for replication from the pSC101 origin. These are defective in DNA binding. Mutations that mapped elsewhere were temperature-sensitive.

The Escherichia coli dnaA gene: four functional domains.

The Escherichia coli DnaA protein is a sequence-specific DNA binding protein that promotes the initiation of replication of the bacterial chromosome, and of several plasmids including pSC101. Twenty-eight novel missense mutations of the E. coli dnaA gene were isolated by selecting for their inability to replicate a derivative of pSC101 when contained in a lambda vector. Characterization of these as well as seven novel nonsense mutations and one in-frame deletion mutation are described here. Results suggest that E. coli DnaA protein contains four functional domains. Mutations that affect residues in the P-loop or Walker A motif thought to be involved in ATP binding identify one domain. The second domain maps to a region near the C terminus and is involved in DNA binding. The function of the third domain that maps near the N terminus is unknown but may be involved in the ability of DnaA protein to oligomerize. Two alleles encoding different truncated gene products retained the ability to promote replication from the pSC101 origin but not oriC, identifying a fourth domain dispensable for replication of pSC101 but essential for replication from the bacterial chromosomal origin, oriC.

Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.

Ordered and sequential binding of DnaA protein to oriC, the chromosomal origin of Escherichia coli.

DnaA protein of Escherichia coli acts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC. By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitro replication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriC in which all DnaA boxes are occupied. The last site bound is DnaA box R3. This event may be critical in promoting initiation from oriC as it correlates with in vivo observations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M. R., Grimwade, J. E., and Leonard, A. C.(1995) EMBO J. 14, 5833-5841).

The A184V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermolability in initiation of Escherichia coli DNA replication.

The temperature-sensitive dnaA5 and dnaA46 alleles each contain two missense mutations. These mutations have been separated and the resulting mutant proteins studied with regard to their role in initiation of DNA replication in vitro. Whereas the His-252 to tyrosine substitution (H252Y) unique to the dnaA46 allele did not affect the activities of DnaA protein, the unique substitution of the dnaA5 allele, Gly-426 to serine (G426S), was reduced in its DNA-binding affinity for oriC, the chromosomal origin. This suggests that the C-terminal region of the DnaA protein is involved in DNA binding. The alanine-to-valine substitution at amino acid 184 (A184V) that is common to both of the alleles is responsible for the thermolabile defect and lag in DNA synthesis of these mutants. Mutant proteins bearing the common substitution were defective in ATP binding and were inactive in a replication system reconstituted with purified proteins. DnaK and GrpE protein activated these mutant proteins for replication and ATP binding; the latter was measured indirectly by the ATP-dependent formation of a trypsin-resistant peptide. However, with this assay, the ATP-binding affinity appeared to be reduced relative to wild-type DnaA protein. Activation was by conversion of a self-aggregate to the monomer, and also by a conformational alteration that correlated with ATP binding.

Novel alleles of the Escherichia coli dnaA gene are defective in replication of pSC101 but not of oriC.

Five novel alleles of the Escherichia coli dnaA gene that were temperature sensitive in maintenance of pSC101, a plasmid that is dependent on this gene for replication, were isolated. Nucleotide sequence analysis revealed that four of the five alleles arose from single base substitutions, whereas the fifth contained three base substitutions, two of which were silent. Whereas all five alleles were temperature sensitive in vivo for pSC101 maintenance, genetic and biochemical characterization indicated that only two were defective in replication from the chromosomal origin, oriC. As previously characterized mutations are defective in replication for both pSC101 and oriC, the dnaA mutations specifically defective in pSC101 maintenance represent a novel class. We speculate that one or more of these pSC101-specific mutants are defective in interaction with pSC101 RepA protein, which is also required for initiation of plasmid DNA replication.

Synthesis of DnaK protein during the division cycle of Escherichia coli.

DnaK protein is involved in the initiation of DNA synthesis from the Escherichia coli chromosome as well as from the replication origins of phage lambda and P1. The synthesis of dnaK mRNA and protein has been reported to vary during the cell cycle of Caulobacter crescentus (Gomes et al., 1990). We have measured the expression of DnaK protein during the E. coli division cycle using the membrane-elution method. Cells labelled with a radioactive amino acid at different times during the division cycle were analysed for radiolabelled DnaK protein by quantitative immunoprecipitation, gel electrophoresis and autoradiography. In contrast to reports of cell-cycle-specific synthesis of DnaK protein in C. crescentus, we find the synthesis of DnaK protein to be invariant during the E. coli division cycle. Its synthesis occurs exponentially, as does the synthesis of total cell protein.

DnaA protein directs the binding of DnaB protein in initiation of DNA replication in Escherichia coli.

DnaA protein of Escherichia coli acts in the initiation of chromosomal replication to bind to sequences in the chromosomal origin. On binding, it promotes the assembly of other replication proteins that serve to prime DNA replication and assemble the replication apparatus for bidirectional replication fork movement. A collection of monoclonal antibodies to DnaA protein have been produced, one of which is described here, that interferes with the action of DnaA protein in promoting formation of a prepriming complex. On the analysis of this process, the antibody appears to interfere with the physical interaction between DnaA and DnaB protein in the DnaB.DnaC complex. Cross-linking studies confirm that DnaA and DnaB proteins interact directly. These results provide the first direct evidence that one of the roles of DnaA protein is to act as a site for binding of DnaB protein to the DNA and perhaps orients DnaB helicase to account for the directionality of replication fork movement.

Open-complex formation by the host initiator, DnaA, at the origin of P1 plasmid replication.

Replication of P1 plasmid requires both the plasmid-specific initiator, RepA, and the host initiator, DnaA. Here we show that DnaA can make the P1 origin reactive to the single-strand specific reagents KMnO4 and mung bean nuclease. Addition of RepA further increased the KMnO4 reactivity of the origin, although RepA alone did not influence the reaction. The increased reactivity implies that the two initiators interact in some way to alter the origin conformation. The KMnO4 reactivity was restricted to one strand of the origin. We suggest that the roles of DnaA in P1 plasmid and bacterial replication are similar: origin opening and loading of the DnaB helicase. The strand-bias in chemical reactivity at the P1 origin most likely indicates that only one of the strands is used for the loading of DnaB, a scenario consistent with the unidirectional replication of the plasmid.

Activation of mutant forms of DnaA protein of Escherichia coli by DnaK and GrpE proteins occurs prior to DNA replication.

Mutant forms of DnaA protein, inert in a replication system composed of other purified proteins, are "activated" by DnaK and GrpE heat shock proteins (Hupp, T. R., and Kaguni, J. M. (1993) J. Biol. Chem. 268, 13137-13142). The effect of these heat shock proteins on DnaA5 and DnaA46 protein was separated from the event of DNA synthesis by incubation in two stages. Components necessary during the first stage for "activation" included GrpE and DnaK proteins, ATP at 0.2 mM or greater, and polyvinyl alcohol (8%) or glycerol, optimal at concentrations between 20 and 30%. An ATP regenerating system provided by creatine kinase and creatine phosphate was stimulatory. Addition of the activated form of DnaA5 or DnaA46 protein to a reconstituted system containing other purified replication proteins during the second stage of incubation resulted in DNA replication. Activation of DnaA5 or DnaA46 protein by heat shock proteins was thermolabile, suggesting that the temperature sensitivity of dnaA5 and dnaA46 mutants is related to this thermolabile interaction. A third heat shock protein, DnaJ protein, interfered with the activation of DnaA5 protein if present during the first stage of incubation. This inhibitory effect was less striking if included during the second stage of incubation. These results suggest a mechanism for regulation of the activity of DnaA protein.

DnaA5 protein is thermolabile in initiation of replication from the chromosomal origin of Escherichia coli.

A mutant form of DnaA protein encoded by the dnaA5 allele has been purified and compared biochemically to its wild type counterpart. Biochemical properties of DnaA5 protein include: 1) thermolabile activity in an oriC plasmid replication system dependent on a crude enzyme fraction, 2) comparable affinity relative to DnaA protein in binding to restriction fragments containing either the Escherichia coli chromosomal origin, oriC, or the dnaA promoter, 3) formation of a nucleoprotein complex similar to DnaA protein at the DnaA boxes within the dnaA promoter as detected by protection from DNase I cleavage, 4) formation of an altered nucleoprotein complex with oriC as judged by DNase I protection experiments, 5) inactivity in unwinding of oriC, 6) inactivity in oriC plasmid replication systems dependent on purified enzymes, and 7) inhibition of DnaA protein by addition of DnaA5 protein in assays of replication and of unwinding of oriC, suggesting that mixed complexes formed between wild type and mutant proteins are inactive.

Activation of DnaA5 protein by GrpE and DnaK heat shock proteins in initiation of DNA replication in Escherichia coli.

DnaA5 protein, inactive in replication systems dependent on purified enzymes, was activated by addition of a crude enzyme fraction. Based on this assay, two proteins in the crude enzyme fraction were identified that confer replication activity upon DnaA5 protein in a purified enzyme system. That one is DnaK protein was suggested initially from studies in which DnaK protein stimulated another mutant form of DnaA protein in replication assays dependent on a crude enzyme fraction (Hwang, D. S., and Kaguni, J. M. (1991) J. Biol. Chem. 266, 7537-7555). The identification of DnaK protein as a required protein for activation of DnaA5 protein, and its inclusion in a reconstituted enzyme system of purified replication proteins, allowed for the partial purification of the second activating protein. GrpE protein was deduced to be the second activating protein based on three criteria. First, activity in partially purified fractions correlated with a protein similar in size to GrpE protein on a sodium dodecyl sulfate-polyacrylamide gel. Second, activity in partially purified fractions correlated with a protein that reacted with anti-GrpE antibody. Third, purified GrpE protein functionally replaced the second protein in activation of DnaA5 protein.