©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of the Initiator Protein DnaA of Escherichia coli with Its DNA Target (*)

(Received for publication, March 15, 1995; and in revised form, May 19, 1995)

Sigrid Schaper , Walter Messer (§)

From the Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin-Dahlem, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Equilibrium and kinetic rate constants were determined for the binding of the initiator protein DnaA of Escherichia coli to its binding site, the non-palindromic 9-bp DnaA box, using gel retardation techniques. The dissociation constant for specific binding was between 1 and 50 nM for individual DnaA boxes on 21-bp double-stranded oligonucleotides. Only DnaA boxes of the sequence TT(A/T)TNCACA resulted in specific fragment retention. Both the 9-bp consensus sequence and flanking sequences determined the binding efficiency. One DnaA monomer was found to bind to a DnaA box and to induce a bend of about 40°.


INTRODUCTION

The initiator protein DnaA of Escherichia coli is central for bacterial replication from the chromosomal origin, oriC. DnaA protein recognizes and binds specifically to nine-base pair consensus sequences, termed DnaA boxes, found four times (DnaA boxes R1-4) in the chromosomal origin(1) . Later a fifth DnaA box (Matsui box) was defined by DNaseI footprinting(2) . DnaA protein binding induces a localized unwinding of AT-rich sequences in the left part of oriC(3, 4) and presumably directs the DnaB/DnaC helicase into this entry site. DnaA acts thereby as a replisome organizer whose interaction with oriC creates the correct structural arrangement for the subsequent loading of proteins required for initiation and replication(5, 6) .

In addition to this structural role DnaA protein can fulfill the combined functions of the primosomal proteins PriA, PriB, PriC, and DnaT in the assembly of a primosome. DnaA priming is the priming mechanism for chromosomal replication (7, 8) and is operative at plasmid origins (9, 10) and in the conversion of single-stranded to double-stranded DNA(11, 12) .

DnaA has been shown to be a transcription factor for various genes. The binding of DnaA to DnaA boxes located within promoter regions leads to repression of transcription of the dnaA gene itself (13, 14, 15, 16) , of the mioC gene, the uvrB gene, and the rpoH gene. DnaA binding results in activation of the glpD gene and the nrd operon (for reviews see (17) and (18) ).

DnaA binds ATP and ADP with high affinity, but only ATP-DnaA is active in oriC replication(19, 20) . ATP and ADP forms of DnaA are monomers in solution,()but the nucleotide-free form tends to form large aggregates(21) . A rapid release of bound ADP and exchange by ATP is catalyzed by phospholipids (22, 23) .

The C-terminal 94 amino acids of DnaA protein, containing a potential helix-loop-helix motif, mediate the specific binding to DnaA boxes (24) . The biochemical details of the binding of DnaA to DNA are, however, not known, and the process is still poorly understood. Binding of proteins to oriC is further complicated due to the presence of additional target sequences for histone-like proteins. The result is the formation of a large nucleoprotein complex(6, 21, 25) whose structure is determined by protein-DNA interactions, protein-protein interactions, and DNA topology (for a recent review see (18) ).

As an approach to define the biochemical details of these interactions we studied the binding of DnaA to single target sites, to DnaA boxes occurring in oriC and within the promoter regions of the mioC and rpoH genes, by determination of equilibrium dissociation constants and kinetic rate constants. We also analyzed structural aspects of DnaA-oriC interaction including the stoichiometry of DnaA molecules per binding site and the induced bending of DNA by DnaA.


MATERIALS AND METHODS

Proteins and DNA

DnaA protein was purified as described (26) except that 100 mM potassium glutamate was used instead of KCl. pBend2-R4/oriC was used for the generation of permuted fragments. It was constructed by insertion of a 21-bp()synthetic oligonucleotide containing DnaA box R4/oriC into SalI/XbaI of pBend2 (27) (the designation ``/oriC'' indicates DnaA boxes flanked on both sides with corresponding sequences from oriC). DNA fragments were purified from agarose gels by cutting out the bands and separating on quick spin columns (Qiagen). Oligonucleotides were synthesized on a DNA synthesizer (Applied Biosystems model 381 A) by the phosphoamidite method and purified by high performance liquid chromatography. Complementary oligonucleotides were annealed by heating at 95 °C and gradually cooling to room temperature. DNA was P-labeled using T4 polynucleotide kinase (Boehringer Mannheim) as described(28) . Isotopes were purchased from Amersham Corp. Groove-binding drugs were from Sigma (distamycin A, 4`,6-diamidino-2-phenylindole), Polysciences (Hoechst 33258) and Molecular Probes (SYBRgreen).

Binding and Gel Retardation Assays

All binding reactions were carried out in 20 µl of binding buffer (20 mM HEPES-KOH, pH 8.0, 5 mM magnesium acetate, 1 mM NaEDTA, 4 mM dithiothreitol, 1 mM ATP, 5 mg/ml bovine serum albumin, 0.2% Triton X-100, and 5% glycerol)(10) . 0.01-2 nM radiolabeled DNA probe and various amounts of DnaA in the nM range were used (indicated in the figures). In some experiments poly(dI-dC) or salmon sperm DNA was added as competitor. Gels were prerun at 8 V/cm for 60 min. Reactions were incubated 10 min on ice and 20 min at 20 °C and applied to 4-8% native polyacrylamide gels running at 11-14 V/cm and 20 °C in TBE buffer (89 mM Tris borate, pH 8.0, 2 mM NaEDTA). After dyes had entered the gel, the voltage was reduced to 9 V/cm. Following electrophoresis the gel was dried and analyzed using a PhosphorImager (Molecular Dynamics). For gel shift experiments with permuted fragments polyacrylamide gels were run continuously at 4-5 V/cm, 4 °C.

Complex Dissociation

Complexes between the purified DnaA protein and the labeled duplex 21-mer oligonucleotides were formed as described above. 50 nM DnaA and 27 nM radiolabeled DNA probe were used. The reaction mixture was allowed to reach equilibrium, and unlabeled DNA of the same oligonucleotide (2.7 µM) was added at time intervals as indicated in Fig. 3A. The complexes were separated by electrophoresis on native 8% polyacrylamide gels running in TBE buffer. Gels were dried and analyzed using a PhosphorImager (see above).


Figure 3: Dissociation of DNA-DnaA complexes. A, 50 nM DnaA protein was incubated with 27 nM radiolabeled duplex oligonucleotides containing the DnaA box TTATCCACA with naturally flanking sequences (R1/oriC and R4/oriC). The protein-DNA complexes were quenched with a 100-fold excess of unlabeled probe for the indicated times (10, 20, 30, 40, and 50 s; 1, 2, 5, and 10 min). The amount of oligonucleotide bound to protein at different times was quantified using a PhosphorImager (Molecular Dynamics). The data were used to plot the results shown in B. B, the time courses for dissociation of protein-DNA complexes between DnaA and duplex oligonucleotides R1/oriC and R4/oriC are shown. t was calculated from the slope. Counts, total cpm in retarded bands as obtained from the analysis with ImageQuant software (Molecular Dynamics).



Stoichiometry of Complexes

The stoichiometry of the complexes (DnaA molecules/binding site) observed in the gels was determined directly in double label experiments using the gel retardation assay. Purified DnaA protein was labeled with N-succinimidyl-[2,3-H]propionate(29) . The P-labeled oligonucleotides (21 bp) or a 105-bp polymerase chain reaction fragment (oriC coordinates +235 to +339; see (30) ) containing DnaA box R4 were complexed with H-labeled DnaA and analyzed by electrophoresis on native 8% polyacrylamide gels. The autoradiograms were used as templates to excise the bands for scintillation counting. Gel slices were oxidized with HO, HClO overnight at 60 °C as described (31) and added to 11 volumes of ReadyValue mixture (Beckman). The amounts of DNA and protein in the complex bands were analyzed by liquid scintillation counting in a Beckman LS 7800 counter. Background was determined by counting of control gel slices from unused regions of the gels.

Pretreatment of DNA with Groove-binding Drugs

2-5 nM radioactively labeled DNA containing DnaA box R4/oriC (105-bp polymerase chain reaction fragment; see above) was preincubated with distamycin A (0.001-50 µM), Hoechst 33258 (0.1-50 µM), 4`,6-diamidino-2-phenylindole (0.1-50 µM), or SYBRgreen (diluted 1:50,000-1:100) for 10 min at room temperature. Then DnaA protein (50 nM) was added and incubated as described under binding assays (see above). In parallel experiments DnaA was omitted from the reaction to study the effects of the ligands alone.


RESULTS

DnaA Binding to Different DnaA Boxes

Purified DnaA protein was investigated for its binding properties with respect to various DnaA boxes occurring in oriC and in promoter regions of selected genes. Synthetic duplex 21-mer oligonucleotides containing the DnaA box or its modified versions as indicated in Table 1were used in gel shift assays. The first eight duplex oligonucleotides contain the DnaA box within averaged flanking sequences. Averaging of flanking sequences was carried out by aligning the four DnaA boxes of the oriC region and selecting the bases by frequency of occurrence. As a control for nonspecific binding a duplex oligonucleotide with a scrambled consensus sequence was included. The second eight oligonucleotides contain the box with the flanking sequences as they occur in oriC (Fig. 1).




Figure 1: DNA binding properties. Gel retardation assays were carried out as described under ``Materials and Methods'' using 2 nM duplex oligonucleotide, 1 µg of poly(dI-dC), and the following concentrations of DnaA protein: without DnaA (lanes1), 50 nM (lanes2), 100 nM (lanes3), and 190 nM (lanes4). Under the conditions used only specific complexes were seen. DnaA boxes with naturally flanking sequences are shown, except for the nonsense box (see Table 1).



The equilibrium dissociation constants K for the protein-DNA interaction were determined by the method of Carey(32) . For this analysis we used a fixed input DNA concentration and various DnaA protein concentrations, spanning at least 4 orders of magnitude (Fig. 2A). The DNA concentration in the reaction mixture was chosen much lower than the protein concentration required for half-maximal binding, so the protein concentration at half-maximal binding is very close to K (Fig. 2B). The K values obtained from this analysis, based on K = [S] [P]/[SP], are shown in Table 1.


Figure 2: Equilibrium binding of DnaA to DnaA box R2. A, 1 nM box R2 with averaged flanking sequences was incubated with the indicated concentrations of DnaA as described under ``Materials and Methods.'' Samples were analyzed by electrophoresis on 8% native polyacrylamide gels, which were dried, and the bands were quantified using a PhosphorImager. The data for other DnaA boxes were obtained identically, except that the input DNA concentration was below 0.2 nM for the boxes R1/oriC and R4/oriC. B, quantitation of the gel assay. Squares show the average of 13 gels (± S.D.). The data for all other DnaA boxes were plotted identically to obtain the values listed in Table 1.



In averaged environment, boxes R1/R4 and R2 were bound specifically by DnaA with K values in the moderate nM range. Unexpectedly, neither box R3 nor the Matsui box nor the P box exhibited specific binding of DnaA. The P box and the ``artificial'' box RY showed similar affinities as R1/R4 and R2. Therefore the fifth position of the consensus sequence is probably not involved in the binding process. Boxes R1 and R4 with naturally flanking sequences showed about 50 times higher affinity, which reveals that the sequences adjacent to DnaA boxes have a significant influence. DnaA interaction with the native P box gave a faint complex band with moderate affinity (Fig. 1).

In contrast, box R3, the Matsui box, and box R4, where all thymidines in the DnaA box had been replaced by deoxyuridine, were not recognized by DnaA specifically. In a parallel experiment hybrids between the natural thymidine containing box R4 and its complementary strand containing three deoxyuridines in the DnaA box were constructed. Both versions of hybrids were not recognized specifically by DnaA, suggesting that both DNA strands participate in the binding reaction (data not shown). Single-stranded DNA containing box R4 was also only recognized in a nonspecific manner. These results corroborate the results reported by Parada and Marians(10) , who demonstrated strong binding to a duplex oligonucleotide containing DnaA box R1/R4 (occurring in pBR322) but only weak interaction between DnaA and single-stranded DNA or RNA/DNA hybrids containing the same box.

K could not be determined precisely from the gel studies because individual complexes were not resolved, but nonspecific binding occurs only at DnaA concentrations above 200 nM. The binding behavior of DnaA in the nucleotide-free form and in the presence of various ribonucleotides like ATP, ADP, ATPS, cAMP, and ATP plus cAMP (1 mM each) to box R2 was studied. None of those had an influence on the dissociation constant (data not shown).

Interestingly, the affinity of DnaA to whole oriC is comparable with the affinity to individual boxes like R1/oriC or R4/oriC. The binding properties were also not affected by in vitro dam methylation of the target DNA, suggesting that methylation does not modulate the binding affinities of DnaA.

Kinetics of Dissociation

Dissociation rates were determined for the complexes between DnaA and the duplex oligonucleotides containing box R1/oriC and R4/oriC. Complexes were formed between labeled DNA and DnaA protein, and after equilibrium was reached an excess of unlabeled oligonucleotide was added. The amount of complex remaining in the reaction was measured as a function of time using the gel retardation assay (Fig. 3A). The amount of oligonucleotide bound by protein at different times was quantified using a PhosphorImager. The time required for half of the complex to dissociate, t, and the dissociation rate constant k were calculated from the data in Fig. 3. The derived values for t and k (Table 2) reveal similar dissociation rates for the binding sites R1 and R4. Dissociation rate constants for DnaA box R2 and association rate constants could not be determined by the gel retardation method, because at the shortest time possible all complexes were already dissociated or associated, respectively.



One DnaA Monomer Combines with One DnaA Box

The DnaA binding unit responsible for recognition at the non-palindromic 9-bp consensus sequence has not been established. Size exclusion chromatography revealed only the monomeric form of DnaA under various conditions: in the absence of ribonucleotides and in the presence of ATP or ADP (data not shown). In order to investigate if DnaA oligomerizes upon binding to individual boxes, complexes between H-labeled DnaA and P-labeled DNA containing a single DnaA box (R4) were formed and separated by gel retardation. Determination of specific activities and quantification of the isolated complexes gave a stoichiometry of 0.8 ± 0.1 per binding site, independent of the length of the DNA. Longer DNA fragments (>100 bp) tended to exhibit higher complexes besides the expected specific complex. The stoichiometry of higher complexes could not be determined by this method due to their faint appearance and inadequate resolution.

DnaA Induces DNA Bending at Its Recognition Site

So far it is not known whether binding of DnaA induces a bend in DNA. We employed the gel shift mobility analysis of permuted fragments (33) to determine whether this is the case for the DnaA boxes R1, R2, and R4 in oriC. A set of permuted fragments was generated from plasmid pBend2-R4/oriC, which contains box R4 as a 21-bp oligonucleotide insertion between two tandemly repeated multicloning sites(27) . Gel retardation of the complexed fragments demonstrates that binding of DnaA to its recognition site introduces a curvature in the DNA (Fig. 4). We have estimated the induced bending angles using the formula derived from an empirical relationship between the electrophoretic mobility retardation and the bending angle(34) . The bending angle resulted in a moderate deviation from linearity of 42 ± 4° for fragments containing box R4/oriC. The uncomplexed fragments ran with identical mobility, indicating the absence of an intrinsic bend in the DNA (Fig. 4). Similar bending angles were observed for fragments containing box R1/oriC or R2/oriC, respectively (data not shown).


Figure 4: Gel retardation analysis of permuted fragments containing DnaA box R4/oriC. Binding reactions and gel retardation were done as described under ``Materials and Methods,'' using 100 ng of DnaA protein. Gel concentration was 6%. DNA fragments were generated by restriction with the enzymes BglII (lanes1 and 6), XhoI (lanes2 and 7), EcoRV (lanes3 and 8), NruI (lanes4 and 9), and BamHI (lanes5 and 10); lanes1-5 show fragments without DnaA.



DnaA Makes Contacts with the Major and the Minor Groove of the Target DNA

The fact that DnaA does not recognize a DnaA box in which thymidines are replaced by deoxyuridines indicates specific contacts with the major groove, because the methyl groups of thymidines are only exposed in the major groove(35) . In order to determine whether DnaA makes contacts with the minor groove as well, we measured the competition of minor groove binding drugs with DnaA binding. Distamycin A binds AT-specifically to the minor groove of B-DNA and is able to remove DNA bends(33, 36) . 4`,6-Diamidino-2-phenylindole and Hoechst 33258 are fluorescent DNA ligands with strong affinity for the minor groove but without structural effects(37, 38, 39) . All three drugs compete with minor groove binding proteins.

DNA fragments containing DnaA box R4/oriC were preincubated with increasing amounts of groove-binding drugs and subsequently allowed to form complexes with DnaA protein. None of the ligands used had an influence on the mobility of the DNA alone, except that at very high concentrations of 4`,6-diamidino-2-phenylindole (>100 µM) and Hoechst 33258 (>10 µM) the DNA tended to be retained in the slots. Formation of the complex with DnaA was impaired by 4`,6-diamidino-2-phenylindole (>10 µM), Hoechst 33258 (>1 µM) and distamycin A (>1 nM). Fig. 5shows the results obtained from DNA preincubation with distamycin A. From these results we conclude that DnaA also makes contacts with the minor groove. Furthermore DnaA protein-DNA interaction was severely inhibited by very low concentrations of SYBRgreen, suspected to bind to the grooves rather than to the backbone of DNA. We conclude that DnaA makes specific contacts to both the major and the minor groove of DNA.


Figure 5: Gel retardation of DnaA box R4/oriC complexes after pretreatment of the DNA with distamycin A. Pretreatment of the DNA, binding reactions, and gel retardation were carried out as described under ``Materials and Methods.'' Lanes1 and 8, DNA only; lanes2-7, DNA pretreated with the drug without DnaA; lane2, 1 nM; lane3, 10 nM; lane4, 100 nM; lane5, 1 µM; lane6, 10 µM; lane7, 50 µM distamycin A; lane9, complexed DNA without drug treatment. Lanes10-16 show the drug-treated DNA with subsequent DnaA complex formation (50 nM DnaA). Lane10, 1 nM; lane11, 10 nM; lane12, 100 nM; lane13, 500 nM; lane14, 1 µM; lane15, 10 µM; lane16, 50 µM distamycin A.




DISCUSSION

Kinetic and equilibrium constants, as well as the stoichiometry and induced DNA bending, were determined for the binding of the initiator protein DnaA of E. coli to its non-palindromic 9-bp binding site using gel retardation techniques. The precise sequence was of primary importance. Specific binding was only observed if the sequence conformed to 5`-TT(A/T)TNCACA. This is the most stringent definition for the DnaA box consensus sequence. More relaxed consensus sequences were previously defined using other techniques. With DNaseI footprinting the consensus sequence for binding site protection was T(T/C)(A/T)T(A/C)CA(C/A)A(1) . An even more relaxed consensus sequence, (T/C)(T/C)(T/A/C)T(A/C)C(A/G)(A/C/T)(A/C), was defined using the ability of the DnaA-DnaA box complex to block in vivo transcribing RNA polymerase(40) .

In addition to the 9-bp DnaA box, sequences flanking the boxes were found to influence the binding affinity. The presence of the six nucleotides from oriC on both sides of DnaA boxes R1 and R4, respectively, enhanced the binding affinity about 50-fold. In the oriC context boxes R1 and R4 showed the highest affinity (10M). Binding to the P box was also improved by the naturally flanking sequences, whereas the binding to the oriC DnaA box R2 was reduced (Table 1). Other factors, e.g. DNA topology, may exert an additional effect on the binding efficiency on larger fragments and in vivo.

DnaA box R3 in oriC was not bound specifically, i.e. the K was >200 nM. This is compatible with previous in vivo methylation protection studies (41) where DnaA boxes R1, R2, and R4, but not box R3, were protected throughout most of the cell cycle. However, DnaA box R3 is protected at higher DnaA concentrations in DNaseI footprinting experiments with DNA fragments (1, 42) as well as with supercoiled DNA templates(6) . Since the cellular concentration of DnaA protein is in the µM range (43) we must assume that other factors modulate DnaA binding to box R3 in vivo.

Stoichiometry measurements showed that DnaA-DnaA box complexes consist of one DnaA monomer per DnaA box. Multiple higher order complexes are only formed on longer DNA fragments. The very low half-times of DnaA-DNA complexes indicate a highly dynamic process in which DnaA protein molecules oscillate between the bound and unbound state.

DnaA protein makes contacts with both the major and the minor groove of DNA. Minor groove contacts are suggested by the negative influence of minor groove-specific ligands on complex formation. The inactivity of DnaA boxes containing deoxyuridine on either strand indicates that both strands participate in the binding reaction and points to major groove contacts.

DnaA protein induces a bend of about 40 ° deviation from linearity at its target site. This must be important for the correct folding of the origin and contributes to the higher order nucleoprotein structure required for the initiation of replication.


FOOTNOTES

*
This work was supported by SFB344/Project B6 of the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-30-8413-1266; Fax: 49-30-8413-1385.

Schaper and Messer, unpublished results.

The abbreviations used are: bp, base pair(s); ATPS, adenosine 5`-O-(thiotriphosphate).


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