(Received for publication, March 15, 1995; and in revised form, May 19, 1995)
From the
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°.
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,
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.
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
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).
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
contrast, box R3, the Matsui box, and box R4
K
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.
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.
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.
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
(10
DnaA box R3 in oriC was not bound
specifically, i.e. the K
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.
(
)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) .
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 Na
EDTA). 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).
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 H
O
, 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.
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).
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.
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).
, 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.
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, ATP
S, 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).
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).
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.
M). 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.
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.
S, adenosine 5`-O-(thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.