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J Biol Chem, Vol. 273, Issue 51, 34255-34262, December 18, 1998


Escherichia coli DnaA Protein
THE N-TERMINAL DOMAIN AND LOADING OF DnaB HELICASE AT THE E. COLI CHROMOSOMAL ORIGIN*

Mark D. SuttonDagger , Kevin M. Carr, Matias Vicente, and Jon M. Kaguni§

From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824-1319

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The initiation of chromosomal DNA replication in all free-living organisms is a two-stage process that first involves assembly of the initiation machinery at specific sequences called the origin of DNA replication (see Ref. 1 for a recent review). Once formed, the initiation complex then assembles proteins that act at the replication fork for semiconservative DNA synthesis. In the yeast Saccharomyces cerevisiae, origin sequences are targeted by the six-member origin recognition complex (ORC).1 ORC in a prereplication complex is then recognized by Cdc6 protein, Cdc45 protein, and minichromosome maintenance proteins (reviewed in Ref. 2). The initiation complex is somehow activated by cyclin-dependent kinases and Cdc7-Dbf4 protein kinase, leading to the import of required proteins at the replication forks and DNA synthesis.

In Escherichia coli, DnaA protein is the functional counterpart to ORC in that it recognizes specific sequences in the replication origin, oriC. Once bound, it directs formation of the initiation complex through a series of discrete steps. First, it induces a local distortion of an AT-rich region near the left boundary of oriC to form an intermediate, the open complex, in an ATP-dependent process that is assisted by either HU or IHF (3-5). The prepriming complex is then formed by the binding of DnaB helicase from the DnaB-DnaC complex (6, 7). Replication fork assembly and DNA replication follow by the coordinated activities of primase and the tau  subunit of DNA polymerase III holoenzyme, each forming contacts with DnaB for concerted primer formation, primer extension, and replication fork movement (8, 9). Processive DNA synthesis also requires single-stranded DNA-binding protein (SSB) and DNA gyrase to relieve positive superhelicity ahead of the replication fork.

In the above sequence of events, the entry of DnaB protein at oriC is an important step in the initiation process because this protein is required for bidirectional replication fork movement. How this occurs is poorly understood and limited to the following observations. First, DnaB protein does not bind efficiently to single-stranded DNA bound by SSB (10). At oriC, DnaA protein induces strand opening in the AT-rich region near the left boundary (3) and also binds to DnaB (11). One model is that DnaA somehow directs the entry of DnaB from the DnaB-DnaC complex to the unwound region covered by SSB so that the helicase is appropriately bound for bidirectional fork movement. Recent UV cross-linking studies have shown that DnaC protein can be covalently coupled to ssDNA (12), suggesting the added participation of DnaC at this step. An important issue is to determine the molecular mechanism whereby DnaB protein enters at oriC to establish the replication forks. This may have relevance to the loading of the replicative helicase at replication origins in the eukaryotic cell. The eukaryotic DnaB counterpart has not been identified.

We recently described a large collection of novel dnaA alleles (13, 14). Their genetic and biochemical characterization indicates four functionally distinct domains. One is a nucleotide binding domain carrying a phosphate binding loop (P-loop) within a predicted secondary structure reminiscent of a Rossmann fold. The second is a region that we speculate is involved in interaction with pSC101-encoded RepA protein. The third is involved in DNA binding. The fourth functional domain is represented by missense mutations clustered near the N terminus. These alleles encode proteins that are inactive in DNA replication. The function of this region until now was uncertain. We previously thought that the region may participate in oligomerization, because missense mutations that reside in this region were active in DNA binding, but were partially defective in transcriptional repression from the dnaA promoter (14). For DnaA protein to autoregulate its expression, the binding of several protomers to DnaA box sequences in the dnaA regulatory region is required that is speculated to involve oligomerization (15). This report examines further the function of the N-terminal domain in initiation of DNA replication. Biochemical characterization of deletion mutants lacking this N-terminal region (DnaADelta 62 and DnaADelta 129 lacking residues 1-62 and 1-129, respectively) revealed that the mutant proteins were active in binding to ATP and to an oriC fragment. The inactivity in DNA replication was due to a defect in forming a replication intermediate termed the prepriming complex. Its formation requires the binding of DnaB from the DnaB-DnaC complex to DnaA protein bound to a DnaA box in a hairpin structure in a single-stranded DNA. The defect was not in the binding to DnaB as measured by surface plasmon resonance. Because several molecules of DnaA protein are bound to a single DnaA box,2 we speculate that DnaA protein monomers assemble at this site to form a unique nucleoprotein structure in which the N-terminal region acts to stabilize DnaB.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Replication Proteins and DNAs-- Monomeric DnaA+, DnaADelta 62, DnaADelta 129 (16), and DnaADelta 219 (17) were purified as described in the respective references from E. coli HMS174(DE3) recA hsdR (rK-12- mK-12+) Rifr (DE3) (F-) (Novagen, Inc.) transformed, respectively, with pKC596 carrying the dnaA+ gene, pKCdnaADelta 62, pKCdnaADelta 129, or pKCdnaADelta 219 (17). The latter plasmids were derived from pKC596 (16) and overproduce truncated forms of DnaA protein lacking the N-terminal 62- (DnaADelta 62), 129- (DnaADelta 129), or 219-amino acid residues (DnaADelta 219). Their purity (>= 95% for DnaADelta 129) and concentration relative to a standard curve derived from bovine serum albumin were determined by scanning densitometry of a Coomassie Blue-stained SDS-polyacrylamide gel using a Gel Doc 1000 unit (Bio-Rad) equipped with the Molecular Analyst software. All preparations of purified DnaADelta 62 contained a smaller polypeptide (~35% by mass based on automated Edman degradation, data not shown) whose N terminus corresponds to residue 89 and whose size is consistent with the C terminus at residue 467 of DnaA protein. The wild type protein is predicted to be 467 amino acids based on DNA sequence (18, 19). The presence of this polypeptide that we presume arose from in vivo proteolysis was taken into account in determining the concentration of DnaADelta 62. Bacillus subtilis DnaA protein was a generous gift from Dr. Shigeki Moriya, Nara Institute of Science and Technology (Nara, Japan). Other replication proteins and DNAs have been described (11, 20).

Surface Plasmon Resonance-- Measurements of the interaction of different forms of DnaA protein with DnaB protein were performed using the BIAcore Biosensor (Biacore AB). DnaB (600 resonance units) was immobilized on a CM5 research-grade sensor chip at 100 µg/ml in 100 mM sodium acetate (pH 4.8) using the carbodiimide covalent linkage method (Biacore AB). A blank surface was prepared by activating and inactivating a sensor chip without any protein immobilization. Measurements were performed at room temperature in 25 mM Hepes-KOH (pH 7.6), 150 mM sodium chloride, 15% glycerol, 1 mM dithiothreitol, and 0.001% Tween 20 with a flow rate of 20 µl/min. All surfaces were washed with 100 mM HCl to remove noncovalently bound proteins.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

N-terminal Deletion Mutants Are Inactive in DNA Replication Activity-- Among a collection of novel dnaA mutations that are defective in DNA replication activity in vivo (13), one class is represented by missense mutations clustered near the N terminus encoding substitutions from amino acids 9-64 (Fig. 1). To understand the function of this region, we examined two N-terminal deletions, DnaADelta 62 and DnaADelta 129 (lacking the N-terminal 62 and 129 residues, respectively), in several in vitro DNA replication assays (Fig. 2). Two assays measured replication activity with an oriC-containing plasmid. In one, required replication proteins were present in a crude protein fraction. In the other, purified replication proteins were used in a reconstituted replication system instead. A third assay measured replication activity with a single-stranded DNA bearing a DnaA box in a hairpin structure and purified replication proteins (21, 22). The mutant proteins were found to be inactive in each of the assays, indicating the essential nature of the N-terminal region (Fig. 2). Also, mixing experiments were performed in which mutant proteins were separately added to reactions and then a subsaturating level of wild type DnaA protein was added. In assays dependent on purified replication proteins, inhibition was observed in proportion to the amount of mutant protein added. However, in the assay dependent on the crude protein fraction, the mutant proteins were marginally inhibitory, possibly because of a protease that degrades the mutant protein.


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  		Fig. 1.   Functional domains of DnaA protein identified by mutational analysis. Symbols (black-triangle) within the functional domains denote the location of each deduced amino acid substitution encoded by the 28 novel missense dnaA alleles (13). Numbers refer to amino acid residues of DnaA protein. Also note that mature DnaA protein is normally post-translationally processed to lack the first residue. The P-loop motif (residues 172-179) is within the Rossmann fold motif (residues 168-235) labeled as "ATP Binding." The end points for respective domains (14) and the secondary structure prediction by the PHD method for the E. coli DnaA protein have been described (14, 51).


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  		Fig. 2.   DnaADelta 62 and DnaADelta 129 are defective in DNA replication. Assays to measure replication activity with the indicated amounts of DnaA+, DnaADelta 62, or DnaADelta 129 were performed with an oriC plasmid (M13oriC2LB5) and purified replication proteins but lacking RNA polymerase, RNase H, and topoisomerase I (panels A and B), with a crude enzyme fraction deficient in DnaA protein (panels C and D) or with M13 A-site ssDNA and purified replication proteins (panels E and F) as described in respective references (11, 52, 53). In panels B and D, the indicated amounts of DnaA+, DnaADelta 62, or DnaADelta 129 were added to reactions then incubated for 5 min on ice prior to addition of 0.6 or 0.8 pmol of DnaA+ protein in the respective panels. In panel F, the indicated amounts of mutant protein were added as above prior to addition of 0.1 pmol of DnaA+. DNA synthesis was then measured as trichloroacetic acid-insoluble radioactivity by liquid scintillation spectrometry.

DnaADelta 129 Is Active in Binding to ATP and to oriC-- Earlier studies identified a phosphate binding loop (P-loop) in DnaA protein that is strictly conserved among 25 dnaA homologs and participates in high affinity ATP binding required for replication activity (16, 23, 24). From a secondary structure prediction algorithm and comparative amino acid sequence analysis, a region in DnaA protein (residues 168-235) is proposed to form a Rossmann fold and also contains conserved sequences that may function to coordinate Mg2+ in Mg2+-ATP (Fig. 1, Ref. 14). This domain is expected to be unaffected by the N-terminal deletions. To demonstrate that the inactivity in DNA replication of the N-terminal truncations was not due to a defect in ATP binding, nitrocellulose filter binding assays were done. We found that the affinity of DnaADelta 129 in binding to ATP (Kd of 0.039 µM) was comparable with that of DnaA+ protein (Kd of 0.02 µM) (data not shown).

Southwestern Analysis of DnaADelta 62 and DnaADelta 129 showed that both bound to a restriction fragment containing oriC with affinities comparable to DnaA+ protein (17). Inasmuch as this method does not accurately measure sequence-specific DNA binding activity, gel mobility shift assays were performed (Fig. 3). DnaADelta 129 formed discrete complexes at comparable levels as DnaA+; with DnaA+ protein, these complexes represent its ordered binding to DnaA boxes of oriC (25). We attribute the slightly faster electrophoretic mobility of DnaADelta 129 complexes compared with DnaA+ complexes to the smaller size of DnaADelta 129. These results indicate that the portion of DnaA protein lacking in these deletion mutants does not affect binding to ATP or to oriC.


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  		Fig. 3.   DnaADelta 129 is active in binding to oriC. Gel mobility shift assays were performed with a 464-base pair SmaI-XhoI oriC-containing DNA fragment derived from pBSoriC as described (25). The relative binding activity in panel B was calculated by quantitative scanning densitometry as the ratio of unbound oriC fragment (free oriC) to total oriC fragment. The inset of panel B shows relative binding at low protein levels.

DnaADelta 62 and DnaADelta 129 Are Active in Unwinding oriC-- DnaA protein has several other distinct activities that contribute to its function in DNA replication. To identify the specific defect of these mutants, assays were performed that measure the formation of replication intermediates. One assay measures the local unwinding of oriC. To address if the N-terminal region functions in this process, linearization of an oriC plasmid by P1 nuclease that cleaves the unwound single-stranded DNA was measured (Fig. 4). In this assay, both mutant proteins were as active as DnaA+ protein. The site of P1 nuclease cleavage was mapped by AflIII restriction to confirm that unwinding was in the AT-rich region (data not shown, Fig. 4 legend). At their highest levels, DnaA+ and DnaADelta 62 were inhibitory but DnaADelta 129 was not. We do not know the reason for this difference.


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  		Fig. 4.   DnaADelta 62 and DnaADelta 129 are active in unwinding of oriC. Unwinding assays (10 µl) were performed essentially as described (3) but contained 25 mM Hepes-KOH (pH 7.6), 1 mM CaCl2, 0.2 mM EDTA, 5 mM ATP, 12% (v/v) glycerol, 0.1 mg/ml bovine serum albumin, M13oriC2LB5 (100 ng), HU (25 ng), and as noted DnaA+ (3, 6, 12.5, 25, 50 ng), DnaADelta 62 (4.3, 8.5, 17, 34 ng) or DnaADelta 129 protein (6, 11, 22, 45 ng). These amounts are listed in picomoles in panel B. After incubation at 38 °C for 15 min, 0.6 unit of P1 nuclease (Amersham Pharmacia Biotech) in 0.01 M sodium acetate (pH 5.3) was added as noted followed by incubation at 38 °C for 15 s. To stop the reaction, 10 µl of 2% SDS and 0.2 M EDTA was added and then samples were incubated at 65 °C for 2 min. Electrophoresis was in 0.7% agarose gels in 90 mM Tris borate buffer. The lane labeled M13oriC2LB5 represents the untreated control to mark the positions of supercoiled and nicked DNA. The position of linear duplex DNA is indicated by the notation "HindIII M13oriC2LB5" (rightmost lane). The relative amount of linear DNA produced by P1 nuclease treatment was determined by quantitative scanning densitometry of the ethidium bromine-stained gel (panel B). To map the sites of cleavage by P1 nuclease, the standard reaction was scaled up 5-fold. The DNA was extracted (GeneClean II kit, Bio 101, Inc. (Vista, CA)) following the manufacturer's protocol, then restricted with AflIII endonuclease, which cleaves the DNA substrate at three sites. Fragments produced confirmed P1 nuclease cleavage in the AT-rich region of oriC (data not shown).

DnaADelta 62 and DnaADelta 129 Are Inactive and Inhibitory in Form 1* Formation-- Because the mutants were active in strand opening of oriC, we next measured the production of a highly negatively supercoiled oriC plasmid termed Form 1* (26). This assay relies on the binding of DnaA protein to DnaA boxes in oriC to result in unwinding of the AT-rich region of oriC (3). Subsequent binding of DnaB protein to this intermediate and its helicase activity enlarges the unpaired region (27, 28). Positive superhelicity induced by more extensive helicase action can be relieved by DNA gyrase to produce Form 1* that is detectable by agarose gel electrophoresis. We found that both deletion mutations were inactive in Form 1* formation, whereas DnaA+ protein was active (Fig. 5). The addition of either DnaADelta 62 or DnaADelta 129 to reactions with DnaA+ protein was inhibitory. This suggests that their inhibitory effect in oriC plasmid replication (Fig. 2, panel B) is due to the formation of mixed inactive complexes with DnaA+ protein.


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  		Fig. 5.   DnaADelta 62 and DnaADelta 129 are inactive in Form 1* formation and inhibit DnaA+ protein. DnaA+ (0.5, 1, 2 pmol) or mutant proteins (0.5, 1, 2, 4 pmol) were added as indicated in panel A to reactions (25 µl) containing M13oriC2LB5 (200 ng), DnaB helicase (90 ng), DnaC protein (25 ng), HU (25 ng), SSB (640 ng), DNA gyrase subunit A (500 ng), and DNA gyrase subunit B (450 ng) as described (29). After incubation at 30 °C for 25 min, products separated by agarose gel electrophoresis were visualized by ethidium bromide staining. The left-most lanes of panels A and B (labeled "M13oriC2LB5") represent the unincubated control. In panels B and C, the indicated amounts of DnaADelta 62 or DnaADelta 129 were added to reactions assembled on ice then incubated for 5 min at 4 °C. DnaA+ protein (1 pmol, where indicated) was then added, and incubation was at 30 °C for 25 min to measure Form 1* formation. Quantitative scanning densitometry of the relative amount of Form 1*, calculated by the reduction in the amount of Form 1 DNA in panel B is shown in panel C.

The activity of the deletion mutants in unwinding of oriC and inactivity in Form 1* formation indicate that the defect is at the step of loading of DnaB at oriC. To consider the exact defect in DnaB loading, one possibility is that because DnaA and DnaB interact physically (11), the deletion mutants are defective in this property. A second is that a specific nucleoprotein complex of DnaA protein bound to DnaA box sequences is required for DnaB to bind to oriC. We could exclude a third possibility that the entry of DnaB at oriC simply involves its binding to the unwound ssDNA that is either naked or covered by SSB. This is because the deletion mutants retained the ability to locally unwind oriC, but were inactive in Form 1* formation.

DnaADelta 62 and DnaADelta 129 Bind to DnaB by Surface Plasmon Resonance-- We showed earlier that DnaA and DnaB physically interact (11), leading to the suggestion that this interaction is important for the delivery of DnaB to oriC. If Form 1* formation requires the direct interaction between DnaA and DnaB for entry of the helicase onto the DNA, the inactivity of the N-terminal deletions in this assay may be due to their inability to bind to DnaB. To measure binding, we used a quantitative method of surface plasmon resonance. This method measures the binding between two (or more) molecules by the change in mass near the sensor surface caused by the binding of one protein from the aqueous phase to a second immobilized on the sensor. This change is measured as resonance units with time after injection of the protein or its removal. With this method, the binding of DnaA protein to immobilized DnaB was in proportion to the amount of DnaA protein injected and showed biphasic kinetics (Fig. 6, panel A). The biphasic kinetics, most likely due to heterogeneity in how DnaB was immobilized to the sensor chip, precludes a kinetic analysis of the data. At similar concentrations, DnaA+, DnaADelta 62, and DnaADelta 129 bound comparably. No binding was seen with DnaADelta 219, a derivative lacking residues 1-219, or DnaA protein from B. subtilis (Fig. 6, panel B). As a control, bovine serum albumin did not bind to DnaB.


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  		Fig. 6.   Interaction of DnaA+, DnaADelta 62, and DnaADelta 129 with DnaB protein. Surface plasmon resonance measurements were performed with a BIAcore Biosensor as described under "Experimental Procedures" with DnaB protein immobilized on the sensor chip. In panel B, the indicated proteins were all at 0.38 µM. For DnaA protein, this corresponds to 20 µg/ml. In panel C, DnaA protein (20 µg/ml, 0.38 µM), or DnaA protein with M7 monoclonal antibody (85 ng/ml) (29), or DnaB (200 µg/ml, 3.8 µM as monomer) were injected. The first downward arrow in each panel indicates the injection of the analyte. The second arrow denotes the onset of buffer flow over the sensor chip. The small reduction in response on injection of DnaADelta 219 was due to the difference in the buffer composition that the protein was in. In each panel, the sample was passaged in series over the sensor bearing DnaB then over a blank surface. The response from the blank surface was subtracted. Surface plasmon resonance measurements were also performed with a C-terminal histidine-tagged derivative of DnaA protein bound to the sensor chip through interaction with an antibody immobilized on the sensor surface (data not shown). The antibody specifically recognizes the histidine6 epitope. Addition of varying amounts of DnaB protein resulted in sensorgrams for which the apparent KD was calculated to be 2 × 10-6 M.

We previously described a monoclonal antibody (M7) that inhibits DnaA protein function by hindering the binding of DnaB from the DnaB-DnaC complex at the step of prepriming (11). This antibody recognizes a conformational epitope within amino acid residues 111-148 (29). As controls, the inclusion of either M7 antibody or DnaB with DnaA protein in the injected sample reduced the binding of wild type DnaA protein to immobilized DnaB (Fig. 6, panel C). These results support the importance of a region from amino acids 111-148 in the interaction with DnaB. However, this interaction is weak (estimated to be in the micromolar range, see Fig. 6 legend), consistent with the requirement for glutaraldehyde cross-linking to stabilize the complex between DnaA and DnaB measured in a solid phase binding assay (enzyme-linked immunosorbent assay) (11). We conclude that the deficiency of the mutants in Form 1* formation is not due to a defect in binding between DnaA and DnaB in the import of DnaB to oriC.

DnaADelta 62 and DnaADelta 129 Are Defective in Prepriming Complex Formation-- Another possible explanation for the inactivity of the N-terminal truncations in the Form 1* assay may be the requirement for a particular nucleoprotein structure formed by the binding of DnaA protein to DnaA box sequences. To test this, we measured the loading of DnaB onto DNA by measuring the ability of the mutant proteins to form a prepriming complex on M13 A-site ssDNA. This single-stranded DNA bears a DnaA box in a hairpin structure (21, 22). We showed previously that DnaA, DnaB, and DnaC are required to form this intermediate that can be isolated by gel filtration chromatography (11). To measure the loading of DnaB onto the ssDNA, DnaB and DnaC protein in the isolated prepriming complex were detected by immunoblotting (Fig. 7). In the negative control involving incubation of proteins in the absence of ssDNA, DnaA+ protein was not detected in void volume fractions (data not shown). The low levels of DnaB and DnaC detected in this control represent the background in this analysis (Fig. 7, first row). In the prepriming intermediate formed with DnaA+ protein and M13 A-site ssDNA, DnaB and DnaC were retained. Side-by-side reactions containing DnaADelta 62 or DnaADelta 129 resulted in void volume fractions with background levels of DnaB and DnaC. In these experiments, ATPgamma S was included to retain DnaC in the prepriming complexes. With ATP instead, its hydrolysis in the DnaB-DnaC complex results in the release of DnaC (6, 7).3 Quantitative analysis to determine the stoichiometry of the proteins in isolated prepriming complexes has confirmed that DnaADelta 62 and DnaADelta 129 are defective in retaining DnaB.2 These quantitative studies showed that in isolated prepriming complexes several molecules of DnaA+, DnaADelta 62, or DnaADelta 129 are bound to a single DnaA box in the ssDNA. We propose that monomers of DnaA protein form a nucleoprotein complex in which the N-terminal region stabilizes DnaB on the SSB-coated ssDNA.


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  		Fig. 7.   Deletion mutants lacking the N-terminal region are defective in loading of DnaB into the prepriming complex. Prepriming complexes were formed and isolated as described (11). Relative to the standard replication reaction, prepriming was scaled up 50-fold in a 100-µl reaction volume in ABC buffer (40 mM Hepes-KOH (pH 8.0), 40 mM potassium glutamate, 4% (w/v) sucrose, 8 mM MgOAc, 0.1 mg/ml bovine serum albumin, 0.1 mM ATPgamma S, and 2 mM dithiothreitol) and contained M13 A-site DNA (5.0 µg) where indicated, DnaB helicase (10 µg), DnaC protein (5.0 µg), SSB (45 µg), and 12 pmol of DnaA+ (625 ng), DnaADelta 129 (460 ng), or DnaADelta 62 (540 ng) as noted. Following incubation at 25 °C for 10 min, prepriming complexes were separated from unbound protein by gel filtration chromatography through Sepharose 4B (Amersham Pharmacia Biotech) equilibrated in ABC buffer. The position of the void volume was determined from the elution of M13 A-site ssDNA that was detected in ethidium bromide-stained agarose gels. DnaB and DnaC protein were identified by immunoblotting of samples (10 µl) from the indicated fractions that were applied to nitrocellulose membranes using a BioDot manifold (Bio-Rad) with affinity-purified rabbit antibody to DnaB or DnaC protein. Detection of immune complexes was with horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad), and the chemiluminescent signal (SuperSignal ULTRA, Pierce) was analyzed with a Bio-Rad GS505 Molecular Imager.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The mechanism of initiation of DNA replication at oriC involves a series of discrete steps. One key event is the entry of DnaB helicase at oriC that results in its proper positioning to advance each replication fork bidirectionally. Once established at each replication fork, DnaB makes contacts with primase in the synthesis of primers for the lagging strand and with the tau  subunit of DNA polymerase III holoenzyme to couple the activity of the helicase to DNA polymerization (8, 9). DnaB binds poorly to duplex DNA or to single-stranded DNA covered by SSB (30, 31). Consequently, a fundamental question is how DnaB is imported onto the DNA at oriC. The experiments described here focus on the role of the N-terminal region of DnaA protein. The functional importance of this region was first appreciated when a large number of novel missense mutations were mapped to amino acid residues from position 9 to 64 (14).

A Functional Domain Near the N Terminus Is Required to Retain DnaB in Forming the Prepriming Complex-- To determine the function of this region, we characterized two N-terminal deletion mutants (DnaADelta 62 and DnaADelta 129 lacking the first 62 and 129 amino acid residues, respectively). The mutant proteins were active in binding to ATP and to a DNA fragment bearing oriC. They were also active in unwinding the AT-rich region at the left end of oriC detected by P1 nuclease cleavage, but were defective in assays measuring different intermediates of the initiation process that each require the loading of DnaB. Notably, DnaADelta 62 and DnaADelta 129 were inactive in Form 1* formation that involves both local unwinding of the AT-rich region of oriC and the entry of DnaB protein so that it can act as a helicase. Because DnaA protein binds directly to DnaB (11), these results suggest that the mutant proteins are defective in this interaction. To test this, experiments measuring prepriming complex formation were done with SSB-coated M13 ssDNA bearing a DnaA box in a hairpin structure. The intermediate formed by the binding of DnaA, DnaB, and DnaC protein is a substrate for primer formation by primase followed by its extension by DNA polymerase III holoenzyme. Compared to DnaA+ protein, immunoblot analysis of isolated prepriming complexes revealed that DnaB was poorly retained by either DnaADelta 62 or DnaADelta 129. Normalizing for the amount of ssDNA, quantitative analysis of isolated prepriming complexes has verified these results.2 Because a similar mechanism is likely to occur at oriC, we conclude that the defect of the N-terminal deletions in Form 1* formation is in promoting the stable binding of DnaB to the prepriming complex. Inasmuch as several molecules of DnaA protein (or N-terminal deletion mutant) are bound per ssDNA,2 this suggests that they form a unique nucleoprotein structure that is required for retention of DnaB.

The N-terminal Domain Is Not Required for Direct Interaction with DnaB-- Contrary to the above results, the defect is not in the binding of DnaA to DnaB per se. By surface plasmon resonance, not only did wild type DnaA protein bind to DnaB but both N-terminal truncations did as well. As controls, binding was reduced by the inclusion of DnaB or a monoclonal antibody (M7) with DnaA protein in the injected sample. This antibody recognizes a conformational epitope within amino acids 111-148 of DnaA protein to inhibit prepriming complex formation (11). These results suggest that two functional domains are involved in the binding of DnaA to DnaB in prepriming. The region of DnaA protein occluded by the antibody is apparently required for the transient (weak) binding of DnaB in its entry into the prepriming complex. In contrast, the N-terminal region lacking in DnaADelta 62 is important for the stable retention of DnaB in the prepriming complex.

Because a number of novel dnaA mutations maps within the N-terminal region lacking in DnaADelta 62, the results presented here suggest that these missense mutants are defective in directing the stable binding of DnaB in the prepriming complex. Likewise, the dnaA508 allele that encodes a substitution of proline at residue 28 (and threonine at position 80) (32-34) is expected to share this defect. We found that a mutant substituted at proline 28 with leucine was active in binding to ATP and to a DNA fragment containing oriC by gel mobility shift analysis, but was inactive in Form 1* activity, suggesting a defect in DnaB loading.4 This interpretation is supported by the observation that B. subtilis DnaA protein failed to bind to DnaB. Comparative amino acid sequence analysis of dnaA homologs reveals that the greatest degree of amino acid sequence variation resides near the N terminus (24).

Unwinding of oriC Is Insufficient for the Loading of DnaB-- The initiation process at oriC involves the unwinding of the AT-rich region near the left boundary that may provide an entry site for DnaB from the DnaB-DnaC complex onto the single stranded DNA. Perhaps the most surprising result of this report is that the N-terminal deletions were active in unwinding the AT-rich region but defective in the loading of DnaB in prepriming complex formation. This was with an M13 derivative carrying a DnaA box in a proposed hairpin structure. Apparently, the ssDNA created by unwinding of oriC or already existing in M13 A-site ssDNA is insufficient for the binding of DnaB. These results indicate that the binding of DnaB to the DNA on which it acts must be mediated by sequences in the N-terminal domain of DnaA. Accordingly, these results support the following model (Fig. 8). First, DnaA protein binds to DnaA boxes in oriC in an ordered manner (25). On binding to ATP, it unwinds a region near the left end of oriC to form the open complex (3, 23). HU or IHF are stimulatory but are not absolutely required at this step (4, 5). Interaction of DnaB protein with DnaA protein, first transiently with amino acids from positions 111 to 148 and then with residues within the first 62 amino acids, results in its stable retention. Release of DnaC protein from the DnaB-DnaC complex unmasks the helicase activity of DnaB. On subsequent unwinding of the parental duplex, the transitory interaction with primase results in the synthesis of primers that are then extended by DNA polymerase III holoenzyme.


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  		Fig. 8.   A model of the initial events at oriC. Sequence elements in oriC include the AT-rich region near the left border and the indicated DnaA boxes. In the first step, DnaA protein binds to respective DnaA boxes in the order indicated numerically (25). ATP-dependent unwinding of the AT-rich region and entry of hexameric DnaB from the DnaB-DnaC complex follows. In the import of DnaB to oriC, DnaB binds to oriC-bound DnaA protein, first transiently with residues within amino acids 111-148, then with the first 62 N-terminal residues (inset). Electron micrographic studies estimate 20-30 molecules of DnaA protein assembled at oriC in the active replication complex with DNA probably wrapped around a central protein core (54-56). Additional DnaA protein monomers (not included for simplicity) interact with those that are already bound to DnaA boxes shown in the lower portion of the model to form the nucleoprotein complex that is active in replication initiation and the loading of DnaB helicase.

How might these results relate to the pathway of initiation in the eukaryotic cell? It is striking that several aspects of the replication process in E. coli and S. cerevisiae are so similar. First, both DnaA and ORC act as sequence-specific DNA binding proteins to target the initiation complex to the replication origin. Second, the binding of these proteins to the replication origin is not sufficient to initiate DNA replication. They are bound at the respective origins at times in the cell cycle when replication does not occur (35-37). In the case of DnaA, the sites bound are DnaA boxes R1, R2, and R4. However, the binding of DnaA protein to DnaA box R3 is critical for initiation to occur (25, 37). Third, processivity of the replicative DNA polymerase (DNA polymerase III core of E. coli or DNA polymerase delta  or DNA polymerase epsilon  in eukaryotic cells) is dependent on a sliding clamp bearing a common structure (beta  subunit of DNA polymerase III holoenzyme in E. coli and proliferating cell nuclear antigen in eukaryotes) and a clamp loader (the DnaX or gamma  complex in E. coli and replication factor C in eukaryotes) (see Ref. 1 for a recent review). Based on these similarities, the finding that DnaA promotes the loading of DnaB at oriC suggests a similar directed mechanism of helicase loading in eukaryotic cells.

Considering that hexameric DnaB is a ring-like structure with the ssDNA passing through the central cavity (38-40), the binding of DnaA to DnaB may result in transient opening of the DnaB ring for ssDNA strand passage and binding to one of the DnaB subunits.

The Entry of DnaB at the lambda  Replication Origin Compared with oriC-- In addition to the requirement for DnaB protein in copying the E. coli genome, many plasmids and phage require dnaB function for replication. In lambda  DNA replication, two lambda -encoded proteins act in the initial events. lambda  O protein is the counterpart to DnaA protein in that it recognizes specific sequences in the lambda  origin (41-43). lambda  P protein forms a complex with DnaB to recruit the helicase to the lambda  origin (44-46) by its binding in the P-DnaB complex to O protein (43, 47, 48). Because P protein and DnaC similarly inhibit DnaB function when complexed to DnaB (45, 46) and O and P protein interact, it has been speculated based on analogy that DnaA and DnaC interact. We showed previously that DnaA interacts with DnaB and not with DnaC by a solid phase binding assay (enzyme-linked immunosorbent assay) (11). These results have been confirmed by surface plasmon resonance measurements.5 With DnaA immobilized on the sensor chip, binding was observed with DnaB and not with DnaC as the analyte. Despite the common requirement for DnaB to function as the replicative helicase in the duplication of the genomes of lambda  and E. coli, the mechanism whereby DnaB is imported to the respective initiation complex appears to be different. Other replicons may have different mechanisms for the entry of DnaB. In the case of R6K, the plasmid-encoded initiator protein (pi) interacts specifically with DnaB (49). In pSC101 replication, genetic evidence suggests an interaction between RepA protein and DnaA, possibly to import DnaB onto the plasmid origin (14, 50)

    ACKNOWLEDGEMENT

We are grateful to Dr. Shigeki Moriya for the gift of B. subtilis DnaA protein.

    FOOTNOTES

* This work was supported by Grant GM33992 from the National Institutes of Health and by Research Excellence Funds from the state of Michigan. This work was also supported by the Michigan Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

§ To whom correspondence should be addressed: Dept. of Biochemistry, Michigan State University, East Lansing, MI 48824-1319. Tel.: 517-353-6721; Fax: 517-353-9334; E-mail: kaguni{at}pilotk.msu.edu.

The abbreviations used are: ORC, origin recognition complex; SSB, single stranded DNA-binding protein; ssDNA, single-stranded DNA; ATPgamma S, adenosine 5'-O-(thiotriphosphate).

2 K. M. Carr and J. M. Kaguni, unpublished results.

3 M. D. Sutton, K. M. Carr, M. Vicente, and J. M. Kaguni, unpublished results.

4 M. D. Sutton and J. M. Kaguni, unpublished results.

5 M. Vicente and J. M. Kaguni, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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