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© 1997 Oxford University Press 3242-3247

From footprint to toeprint: a close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA

From footprint to toeprint: a close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA Christian Speck, Christoph Weigel and Walter Messer*

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

Received May 15, 1997; Revised and Accepted June 30, 1997

ABSTRACT

The Escherichia coli DnaA protein binds as a monomer to the DnaA box, a 9 bp consensus DNA sequence: 54-TTA/TTNCACA. To assess the contribution of individual bases to protein binding we probed the DnaA-DnaA box complex with the uracil-DNA glycosylase (UDG) footprinting technique. (i) dU at the positions of T2, T4, T74 or T94 completely inhibits DnaA binding to the DnaA box. At these positions the methyl groups of the thymine residues are essential for successful DnaA binding, indicating protein contact with the major groove. Additionally they are positioned exactly on one side of the helix. (ii) dU at the position of T1 or at three T residues adjacent to the 9 bp core sequence of the DnaA box allows DnaA binding. These positions are protected from UDG digestion as revealed by the footprint assay. (iii) dU at the position of T34 on the complementary strand of the box 54-TTATCCACA was not protected from UDG digestion in DNA-DnaA complexes. Therefore, DnaA cannot contact the major groove at this position. In addition, a slight bend of the DnaA box towards UDG would help the enzyme to access this site.

INTRODUCTION

The DnaA protein is essential for the oriC-dependent initiation of chromosome replication in Escherichia coli and other bacteria. It binds specifically to an asymmetric 9 bp consensus sequence, the DnaA box, which is present five times in the chromosomal origin (1-3). DnaA has been shown to act as a transcription factor for various genes (4). Binding of DnaA to DnaA boxes in promoter regions leads to repression of transcription as found in the dnaA gene itself, the mioC gene, the uvrB gene and the rpoH gene. Activation is reported for the glpD gene and the nrd operon (5). The C-terminal domain of the DnaA protein is responsible for binding to DnaA boxes (6) and it contains a new binding motif.

The biochemical details of the DnaA-DNA interactions within a single target site (7) have been studied extensively using DNase I footprinting, gel retardation and related techniques. However, little is known about the importance of individual bases in the DnaA box. In order to extend the limits of resolution we adapted a novel technique, UDG (uracil-DNA glycosylase) footprinting, to study the DnaA-DnaA box complex.

The DNA repair enzyme UDG can be used to probe protein-DNA interactions (8): (i) UDG protection (`footprinting'), which measures the ability of UDG to excise uracil residues from a binding site in the presence of binding protein; (ii) missing thymine methyl site (MTM-site) interference assay to identify interactions involving the methyl group of the thymine base (9). For that a bandshift reaction is performed on the mixture of protein and uracil-containing DNA. Residues at which the thymine methyl group is crucial for the complex formation will be absent from the shifted fraction but will be enriched in the free DNA fraction. Therefore this technique allows the determination of which thymine bases in the DnaA box are protected, and how complex formation is influenced by missing methyl groups. In addition, UDG results in a smaller footprint in comparison to DNase I footprints and this footprint method shows, alongside protected regions, a ladder of thymine residues as an inherent size standard (8).

The most stringent definition of the 9 bp consensus sequence of DnaA boxes 54-TTA/TTNCACA (7), comes from a determination of binding constants. DNase I footprint analysis defines 54-TT/CA/T TA/CCAC/AA as the site for specific binding (1,2,10). A still more relaxed DnaA box consensus sequence was found in an in vivo analysis of the effects of DnaA on transcription termination: 54-T/CT/CT/A/CTA/CCA/GA/C/TA/C (11). However, binding affinities vary significantly among the motifs and depend as well on the sequence context.

To study the DnaA-DnaA box complex we choose the single DnaA box in the dnaA promoter region between dnaAp1 and dnaAp2 (12). The DnaA box in the dnaA promoter region fulfils the sequence requirements of the stringent definition: 54-TTATCCACA.

MATERIALS AND METHODS

Bacterial strains and plasmids

Escherichia coli hosts for plasmid propagation were WM1963 [=XL1-Blue (13)] [endA1, gyrA96, hsdR17, recA1, relA1, supE44, thi; F4lac: lacIQ, DlacZM15, proA+, proB+, Tn10 (tetR)] and WM1771 [=RZ1932 (14)] [dut ung Tn10 (tetR); F4 lysA]. Strains were grown in liquid or on solid L-medium at 37_C; L-medium with 50 mg/ml ampicillin was used for transformed strains. Plasmid pDOC170 was the source for DNA restriction fragments. This plasmid contains a 1828 bp PCR fragment with the functional dnaA gene including the entire promoter region (position 578-2404, GenBank accession no. J01602) cloned into the SalI/SacI sites of pOC170 (6), which contains the ColE1 rop replication origin of pBR322 on a NotI cassette, the bla gene of pT7-7 for selection and the chromosomal oriC region of E.coli.

Enzymes, proteins and DNA marker

UDG and 1 kb ladder marker were obtained from Gibco BRL (Bethesda, MD, USA). Restriction enzymes and Klenow fragment were from Boehringer (Mannheim, Germany) or New England Biolabs (Beverly, MA, USA) and used following the manufacturer's instructions. DnaA protein from E.coli was purified as described (7).

Detection instruments and software

PhosphorImager, FluorImager 575, Personal Densitiometer and ImageQuant NT/3.3 software for image processing were from Molecular Dynamics (Sunnyvale, CA, USA).

DNA restriction fragment purification

By choosing a suitable restriction site fragments can be 34-end- labelled with dCTP at one end only. Therefore two different restriction digests of pDOC170 were used for strand-specific detection of footprinting products: BssHII and EcoRI (372 bp) for the upper strand, EcoRV and RcaI (454 bp) for the lower strand. The restriction products were separated by agarose gel electrophoresis (1.5%, 0.5y TBE, 8 V/cm) and the desired fragments collected onto DEAE membrane (NA45, Schleicher & Schuell, Dassel, Germany). The membrane was washed with electrophoresis buffer and the DNA eluted by incubation for 20 min at 65_C in 100 ml NET buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1.25 M NaCl). The DNA was diluted 3-fold with H2O, extracted once with phenol/chloroform (1:1), and precipitated for 30 min at -70_C with 2 vol ethanol using mussel glycogen (Boehringer, Mannheim, Germany) as carrier. The precipitate was collected by high-speed centrifugation, and the pellet air-dried. The pellet was resuspended in 1y TE and adjusted to a final DNA concentration of 25 ng/ml.

UDG footprinting

Aliquots of DnaA were thawed and kept on ice, adjusted to a final concentration of 200 mM ATP (pH 7.0), and diluted into binding buffer [20 mM HEPES-KOH pH 8.0, 5 mM Mg-acetate, 1 mM EDTA, 4 mM DTT, 0.2% Triton X-100, 5 mg/ml bovine serum albumin (Sigma, St Louis, MO, USA), 5% glycerol, 100 mM ATP (15)]. Assay mixtures (usually 10 ml) were obtained by adding the desired protein dilution to 25 ng DNA in H2O on ice. Binding of DnaA to DNA was achieved by incubation of the reaction at 37_C for 5 min. UDG (1 U/ml) was diluted 1:20 in 0.5y binding buffer, 1 ml of the UDG dilution added to the reaction, and the incubation continued for 5 min. Electrophoresis was carried out on 1.5% agarose gels at room temperature in 0.5y TBE buffer (22.5 mM Tris-borate, 0.5 mM EDTA, pH 8.0) at 4 V/cm. Gels were stained with ethidium bromide in electrophoresis buffer (Fig. 1). For the UDG protection assay the desired bands of the `free' DNA and `bound' DNA (complexed with DnaA) were eluted as described above. In the case of the MTM-site interference assay the DNA pellets of the `bound' band were resuspended in 10 ml binding buffer and treated with 1 ml of the UDG dilution for 5 min at 37_C prior to end-labelling.


Figure 1. Band shift assay of DnaA protein with a 372 bp EcoRV-RcaI fragment (25 ng), containing the DnaA box of the dnaA promoter isolated from a dut ung strain. Binding of DnaA and gel electrophoresis were performed as described in Materials and Methods. Gel concentration was 2%. Lane 1, without protein; lane 2, molar ratio of protein:DNA 1:1 (5.4 ng DnaA protein-25 ng DNA; 10 ml volume); lane 3, 4:1; lane 4, 10:1; lane 5, 20:1; lane 6, 1 kb ladder.

Labelling was performed after gel retardation. 34-end-labelling of the DNAs was carried out in 20 ml reactions in 1y TA buffer (33 mM Tris-acetate pH 7.8, 66 mM K-acetate, 10 mM Mg-acetete, 0.5 mM DTT) with 5 mCi [a-32P]dCTP (3000 Ci/mmol, Amersham, Little Chalfont, UK) using E.coli DNA polymerase I (Klenow fragment). Non-incorporated nucleotides were removed by two successive ethanol precipitation steps. For alkali cleavage, the DNA pellets were resuspended in 3 ml 50 mM NaOH, 5 mM EDTA and incubated for 5 min at 95_C. The DNAs were vacuum-dried, resuspended in 5 ml formamide loading buffer, denatured by heat treatment for 5 min at 90_C, and electrophoresed on 6% sequencing gels. Following electrophoresis the gels were fixed in 1% acetic acid and vacuum-dried. The dried gels were exposed to a sensitive screen and evaluated using a PhosphoImager and the ImageQuant software. Alternatively, conventional autoradiograms were obtained by exposing the dried gels to XOmat-AR films (Kodak, Rochester, NY, USA).

T-ladder

The same DNA restriction fragments analysed by footprinting were used to obtain a `T-ladder' size marker for the sequencing gels. The respective fragment (5 ng) was incubated in 8 ml of binding buffer for 5 min at 37_C. An aliquot of 1 ml UDG (1:20 dilution in binding buffer) was added and the incubation continued for further 5 min. Labelling and alkali treatment were performed as described above.

Bromo-deoxyuracil crosslinking assay

Crosslinking was carried out with two pairs of complementary oligodeoxyribonucleotides with the sequences 54-ACAGAGTTATCCACAGTAGAT and 54-ATCTACTGTGGATAACTCTGT, respectively. The sequence of the DnaA box R4 from oriC is shown in bold letters, underlining indicates thymine residues substituted by 5-bromo-deoxyuracil (BdU) in one strand of an oligonucleotide pair which in addition carried a fluorescein label at the 54 end for detection. Annealing of the oligonucleotides in all four possible combinations was carried out as described (13). An aliquot of 40 ng of the annealed double-stranded oligonucleotide in binding buffer was mixed in the wells of a microtiter plate with 300 ng DnaA in binding buffer to give a final reaction volume of 10 ml. Protein binding to the oligonucleotides was achieved by 15 min incubation of the reaction mix at room temperature. For crosslinking the samples were placed on ice and irradiated for 5 min with a germicidal lamp (G8T5 type, Sylvania, USA) at a distance of 5 cm. Subsequently, the samples were split and analysed for protein crosslinked with the fluorescein-labelled and BdU-substituted oligonucleotide by SDS-PAGE (13), while the efficiency of DnaA binding to the oligonucleotides was analysed by a band-shift assay. The band-shift assay was carried out by electrophoresis of the samples on a 2% agarose gel at 4_C in 0.5y TBE buffer at 4 V/cm. Following electrophoresis, both types of gels were read immediately with the FluorImager to detect the fluorescein label. For the detection of unlabeled oligonucleotides, the agarose gels were subsequently stained with SYBR-GREEN (Molecular Probes, Eugene, OR, USA) in electrophoresis buffer according to the manufacturer's instructions. Stained agarose gels were read with the FluorImager. Coomassie blue-stained SDS gels were read with a `Personal Densitometer' device.

RESULTS

The UDG footprinting technique requires deoxyuracil containing DNA (dU)DNA as template. A PCR-based method to generate DNA fragments containing defined amounts of dU was described (8). Alternatively, (dU)DNA could be prepared from a dut ung mutant E.coli strain (14). The dnaA promoter region contains six GATC sites close to the DnaA box. To obtain a template resembling the `natural' DNA structurally as closely as possible, dU-containing PCR fragments would require treatment with Dam methyltransferase whereas (dU)DNA synthesised in vivo in a dam+ host could be used directly.

In order to analyse whether the dU content of DNA prepared from a dut ung strain is sufficient for UDG footprinting, plasmid pDOC170 DNA was prepared by the alkaline lysis method (16) from strain WM1771 (dut ung) and from strain WM1963 as control. The dU-containing plasmid DNA was digested similarly with most restriction endonucleases as the control DNA, and the migration of restriction fragments on agarose gels was normal. Not surprisingly, however, the (dU)DNA could not be completely digested by DraI (TTTAAA) and SspI (ATTAAT). On native polyacrylamide gels, dU-containing restriction fragments from pDOC170 showed a slightly higher electrophoretic mobility, indicating a moderate deviation from standard DNA. By comparing the aberrant mobility of (dU)DNA synthesised in vivo with that of PCR-generated DNA with known dU content, we estimate the content of the former in the range of 1-2% (not shown). The (dU)DNA was as resistant to alkali treatment and heat denaturation as the control DNA. UDG treatment rendered (dU)DNA susceptible to alkali cleavage at apyrimidinic sites resulting in a smear pattern on agarose gels (not shown).

No differences were detectable in band-shift assays for DnaA binding to (dU)DNA as compared to normal DNA. In addition-and most importantly-dU incorporation in vivo was efficient enough and sufficiently random to result, after UDG treatment, in a T-ladder with very little indication of preferred dU incorporation sites or preferred sites of UDG cleavage, respectively (Fig. 2, lanes 1 and 5). We conclude from these experiments that dU-containing DNA prepared from a dut ung strain is an excellent template for the UDG footprinting technique.


Figure 2. UDG footprints of 372 bp EcoRV-RcaI (lower strand, lanes 1-4) and 454 bp EcoRI-BssHII (upper strand, lanes 5-8) dnaA promoter fragments. T-ladder, UDG protection assay and MTM interference assay were done as described in Materials and Methods. Lanes 1 and 5: T-ladder (without protein); lanes 2 and 6: free DNA isolated out of a retardation gel; lanes 3 and 7: bound DNA (first complex) of MTM interference assay, two times UDG treated DNA-first during binding reaction, second after isolation from the retardation gel; lanes 4 and 8: bound DNA (first complex) of UDG protection assay. Bars show the 9 bp DnaA box.

Band-shift assay

In order to analyse DNA bound to protein and free DNA individually, we separated restriction fragments by gel retardation on agarose gels (Fig. 1). The band-shift patterns obtained with (dU)DNA were indistinguishable from those obtained with normal DNA, even when the DNA-protein complexes were treated with UDG prior to gel electrophoresis. The amounts of DnaA protein were adjusted to obtain essentially a 1:1 distribution between protein-bound DNA and free DNA. After complete separation of free DNA and protein-bound DNA, the corresponding bands were excised from the gel and purified as described in Materials and Methods.

UDG analysis

For footprinting the DNA was incubated with DnaA protein and then treated with UDG. Complexes were separated from `free' DNA on an agarose gel. Then the `free' DNA and the first complex were purified. The `missing thymine-methyl interference assay' (MTM interference assay) (9) includes in contrast to the UDG protection assay a second UDG treatment prior to strand-specific labelling of the fragments, alkali cleavage and gel electrophoresis under denaturating conditions. The results of both assays for each strand of the DnaA box region in the dnaA promoter are shown in Figure 2. An enlarged version of the DnaA box region from the original gels is shown together with the schematic interpretation of the results in Figure 3. As mentioned above, UDG treatment of DNA fragments gave clear T-ladders for each strand which allowed unambiguous assignment of protected residues in the actual footprinting experiment.


Figure 3. UDG footprints of the DnaA protein-DnaA box complex and their interpretation. Lanes 1-4 (lower strand), EcoRV-RcaI fragment; lanes 5-12, graphical summary; lanes 13-16 (upper strand), EcoRI-BssHII fragment; lanes 1, 5, 12 and 16, T-ladder; lanes 2, 6, 11 and 15, free DNA; lanes 3, 7, 10 and 14, 2y UDG treated DNA, MTM-site interference assay; lanes 4, 8, 9 and 13, bound DNA, UDG protection assay. Broad black boxes mark the residues whose methyl groups are essential for protein-DNA complex formation. Open boxes indicate protection from UDG. Black boxes mark residues that are cut out by UDG. Lines: not visible in UDG footprint.

UDG protection assay

Missing signals in the UDG footprint of the DNA-protein complex fraction indicate that either bound protein protects the base from UDG digestion, or that dU at these positions prevents protein binding. We found protection of T residues (uracil bases) within the binding site of DnaA protein (DnaA box) for both strands: T1, T2, T4, T74 and T94. There were also three additional protected T residues in a distance up to three bases from the DnaA box. A signal of comparable strength was found for T34 in all lanes. We take this as an indication that this position is not protected from UDG digestion by DnaA binding to the DnaA box.

MTM interference assay

The MTM assay can in contrast to the UDG protection assay discriminate between simple protection of the DNA by DnaA protein and preventing complex formation due to uracil. In the MTM assay the reappearance of signals after the second UDG treatment indicates bases that are protected by DnaA but not essential for binding. The base at position T1 and the preceding base of the upper strand indicate such a protection from UDG digestion by bound DnaA. For the lower strand, signals reappeared at T residues flanking the DnaA box on both sides. Therefore DnaA binding does not require the methyl groups at these positions. Protection is probably due to a sterical hindrance of UDG cleavage by bound DnaA protein.

The signals corresponding to T2 and T4 on the upper strand and T74 and T94 on the lower strand were significantly stronger in the `free' fraction than in the corresponding T-ladders (Fig. 3). Obviously, deoxyuracil bases at one of these positions prevent DnaA binding and lead to an enrichment of fragments containing dU at these positions in the `free' fraction. This result strongly suggests that DnaA binding to the DnaA box requires protein contacts to the methyl groups of T2, T4, T74 and T94 , which are exposed towards the major groove.

Bromo-deoxyuracil crosslinking assay

In a complementary approach we analysed the strand specificity of DnaA binding to the essential T residues of the DnaA box by crosslinking DnaA to 21mer double-stranded oligonucleotides. The oligonucleotides contained the DnaA box R4 from oriC in the wild-type sequence context (7); DnaA box residues T2, T4 on the upper strand, and T74, T94 on the lower strand were substituted by 5-bromo-deoxyuracil. In addition to its ability to form crosslinks with protein upon UV irradiation, 5-bromo-deoxyuracil closely resembles thymine structurally and can replace thymine in vivo (17), and thus should allow DnaA binding. Complementary strands of the substituted oligonucleotides were hybridised to each other or to unsubstituted homologues to allow the strand-specific detection of crosslinked protein. The substituted oligonucleotides were covalently linked at their 54 ends to fluorescein for rapid and sensitive detection of crosslinked material. DnaA was bound to the double-stranded oligonucleotides under standard conditions at a protein/oligonucleotide ratio of 2:1 and irradiated with UV. The samples were then split and analysed separately by a band-shift assay for successful DnaA binding, and by SDS-PAGE for the amount of crosslinked material. The results are shown in Figure 4.


Figure 4. Crosslinking of DnaA to the DnaA box. Reaction mixes of 10 ml each containing 40 ng oligonucleotide were set up as described in Materials and Methods. Forty ng oligonucleotide were also used as size marker in lane 1. The combinations of annealed upper strand and lower strand of DnaA box R4 are indicated by boxes in the top part of the figure: 5-bromo-deoxyuracil substituted, 54 fluorescein-labelled strands are shown as dark grey boxes, while unsubstituted, unlabelled strands are shown as open boxes. For lanes 11-13, 20 ng of fully substituted, labelled oligonucleotide were mixed with 20 ng unsubstituted oligonucleotide in order to have the same amount of fluorescein label in all reactions. Reaction mixes contained 300 ng DnaA giving a DnaA/oligonucleo- tide ratio of ~2:1. 300 ng DnaA were used as size marker in lane 14. A 50-fold excess of unsubstituted oligonucleotides (2 mg) was included as competitor in the samples shown in lanes 4, 7, 10 and 13. Samples in lanes 3, 4, 6, 7, 9, 10, 12 and 13 were irradiated with UV light as described in Materials and Methods. (A and B) show the result of the band shift assay on a 2% agarose gel; (A) shows the fluorescein detection, (B) shows the SYBR-GREEN stain of the region of the same gel containing protein-DNA complexes, scanned at lower sensitivity. (C and D) show the analysis of crosslinks by SDS-PAGE (10% gel). They are derived from the same gel. (C) Fluorescein detection, (D) Coomassie stain. (D) Displays the region of the complex of (C), and indicates that in every lane the same amount of protein is used. Unbound oligonucleotides are marked with `oligo', protein-DNA complexes (and free DnaA protein) are marked with `complex'.

Binding of DnaA to the substituted oligonucleotides was as efficient as binding to the unsubstituted homologues, and not affected by the UV treatment (Fig. 4B, compare lanes 2 and 3 with lanes 5,6, 8,9 and 11,12 respectively). Crosslinks between DnaA protein and either strand of the DnaA box were readily obtained (Fig. 4C, lanes 6 and 9). This indicates that amino acid residues of DnaA protein are in close proximity of the bromine atoms and are therefore most likely involved in protein-DNA contacts. Crosslinking was slightly more efficient for the upper strand than for the lower strand. Crosslinking was specific to the DNA binding site of DnaA protein because it was significantly quenched in the presence of a 50-fold excess of unsubstituted double-stranded oligonucleotide (Fig. 4A and C, lanes 7, 10 and 13). A certain degree of protein degradation due to the UV treatment of the samples was visible in the sensitive fluorescein detection but could not be detected in the Coomassie stain of the same gel (Fig. 4C and D).

DISCUSSION

The interaction of DnaA protein with the DnaA box was analysed with the UDG footprinting technique and the results have been confirmed by crosslinking experiments using 5-bromo-deoxyuracil. Specifically, we asked which thymine bases in a DnaA box are required for complex formation and what can we learn from this about the complex.

The definition for the DnaA box depends on the technique used: 54-TTA/TTNCACA (7), 54-TT/CA/TTA/CCAC/AA (1,2,10) or 54-T/CT/CT/A/CTA/CCA/GA/C/TA/C (11). Additionally the efficiency for DnaA binding is influenced by adjacent sequences (7). However only limited information is available about the possible structure of the DNA-protein complex. DMS footprints showed protection of 34-AATAGGTGT-54 and a sensitive site at 34-AATAGGTGT-54 , which suggests major groove contacts (18). DNase I footprints showed that the full box except for a base in the middle is protected (1). This is also reflected in the definition of the stringent consensus sequence, where the middle base is irrelevant for binding (7).

The UDG footprinting technique permits the study of protein-DNA interactions that involve the thymine bases, which is the most frequent base in the consensus sequence and has not been studied so far. The UDG has the advantage of preserving the sugar-phosphate backbone structure of the DNA during the footprint reaction. Consequently it is possible to separate DNA-protein complexes and free DNA. This is of special importance in the case of DnaA protein, because it tends to form higher order complexes due to protein-protein interaction, as seen in gel retardation. Therefore by contrasting UDG with DNase I footprints, it is possible to study a well defined complex.

The substrate for the enzyme we used for this method was uracil containing DNA from an E.coli dut ung strain. This DNA is Dam methylated in contrast to PCR generated DNA used previously (8) and has proven to be an ideal substrate for the UDG footprinting technique. The percentage of dU incorporation is sufficiently high to give a good footprint and is mostly random (Fig. 2). To study a single DnaA box in a complex with DnaA protein in an invariant stoichiometry, we choose the box in the promoter region of the dnaA gene and purified the first complex from a retardation gel. Three different kinds of DnaA protein-DnaA box interactions could be distinguished.

(i) At the positions T2,T4,T74 and T94 of the DnaA box the methyl groups of thymines are required to permit DnaA protein binding. These sites are positioned in a special way (Fig. 5). The bases are opposing each other pairwise in one turn of the major groove, which could enable an interaction with one or two helices of the DNA binding domain of DnaA protein. This induces a conformational change in DNA seen as a bend (7). Methyl groups essential for complex formation should be in a region of close protein-DNA contact. The successful 5-bromo-deoxyuracil crosslinking of these bases proves that these close contacts are formed. This makes it unlikely that the loss of binding to dU containing DnaA boxes is due to a distortion of the helix.


Figure 5. Model of a DnaA box. The methyl groups of the thymine bases, which are important for the complex, are displayed by balls. Black balls represent methyl groups that are essential for binding. Substitution of these methyl groups by bromine (5-bromo-deoxyuracil) allows crosslinking to DnaA. At positions of grey balls the missing group has no influence on complex formation but DnaA protein protects these bases. The white ball represents a methyl group in the middle of the DnaA box that is not protected by DnaA protein.

(ii) On the lower strand of the DnaA Box one base in the middle, T34 , was not protected. Data from the crystal structure of UDG allows us to define the space this enzyme needs for cleaving the uracil (19). Correlating these data we can conclude that the DnaA protein cannot bind at the positions 34-AATAGGTGT-54 on the lower strand in the major groove. This is corroborated by the results of DNase I footprints, showing an accessible site in the middle of the lower strand of R4 and R2 (1). We conclude that the DnaA protein binds only from one side, from the right in the side view of the DnaA box in Figure 5. A gentle bending of the DNA towards UDG helps the enzyme (19). Therefore the bend induced by DnaA protein is likely to be favorable for excision of uracil. The action of UDG does not destroy the interaction between the DNA and the DnaA protein.

(iii) On both strands uracil bases are close to the DnaA box sequence, which are protected from UDG digestion by DnaA protein. Uracil bases instead of thymines at these positions do not prevent DnaA binding. Therefore protein-DNA interaction at these sites does not include methyl group contacts; presumably this protection is a simple physical obstruction. For the flanking sequence of the DnaA box it is impossible to define a consensus sequence, but there is a strong influence on the efficiency of binding (7). This effect can most likely be explained by the DNA structure of that sequence.

Technical advantages of UDG footprinting

UDG is a small enzyme, and one can define precisely the space the enzyme needs to access the DNA. This helps the interpretation of protection patterns. It is possible to discriminate between bases required for binding and those that are merely covered by a binding protein. The UDG reaction on naked dU-containing DNA provides an exact size marker, a T-lane, which overcomes problems sometimes observed when pinpointing protected regions to the DNA sequence with DNase I footprinting. Because of the precision of UDG footprints we like to call them `toeprints'.

ACKNOWLEDGEMENTS

We thank Andrea Schmidt for purified DnaA protein and excellent technical assistance. We thank Richard Reinhardt and the service group of the institute for the synthesis of the oligonucleotides. This work was supported in part by grant SFB344/A9 of the Deutsche Forschungsgemeinschaft.

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*To whom correspondence should be addressed. Tel: +49 30 8413 1266; Fax: +49 30 8413 1385; Email: messer{at}mpimg-berlin-dahlem.mpg.de
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