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INTRODUCTION |
The initiator protein DnaA plays an essential role in the
initiation of bacterial chromosome replication. The interaction of DnaA
protein with its chromosomal origin (oriC) is best
understood in Escherichia coli. The E. coli DnaA
protein (52 kDa) binds to five nonpalindromic, nonamer sequences, the
DnaA boxes. Binding of 10-20 DnaA monomers promotes a local unwinding
of an adjacent AT-rich region. The unwound region provides an entry
site for the DnaB/DnaC helicase complex followed by other proteins
required to form a replication fork (1-4).
Apart from its primary function as a replisome organizer, the DnaA
protein acts as a transcription factor that represses or activates
several genes or terminates transcription, depending on the location
and arrangement of DnaA boxes (5).
Both functions of the DnaA protein, replisome organizer and
transcription factor, are mediated by its interaction with target DNA.
The DNA binding domain of E. coli DnaA has been localized in
the 94 C-terminal amino acids. A potential helix-loop-helix motif has
been reported within this part of the protein (6). However, because
x-ray high resolution structure analysis is not yet available for DnaA
proteins, the detailed interaction with the DNA is still poorly
understood. The consensus sequence of the E. coli DnaA box
differs depending on the method used for its evaluation. The most
stringent definition for the DnaA box sequence comes from a
determination of binding constants: 5'-TT(A/T)TNCACA-3' (7).
T2, T4, T7', and T9'
were found to be directly involved in DNA-protein interaction (8).
E. coli DnaA does not dimerize in solution and interacts
with a single DnaA box as a monomer, as measured by gel retardation (7)
and by surface plasmon resonance (9). However, binding to DnaA boxes
that differ from the stringent consensus sequence by one or two base pairs requires two such boxes and an interaction of DnaA proteins bound
to them (9). DnaA binds ATP and ADP with high affinity. Both forms of
DnaA protein, ATP-DnaA and ADP-DnaA, recognize DNA in a similar
fashion; however, only ATP-DnaA is active in the DNA replication
process. Recently, it has been shown that E. coli ATP-DnaA
protein recognizes also a hexamer sequence, the ATP-DnaA box
5'-AGATCT-3' or close match of it (9).
Streptomycetes (Gram-positive soil bacteria) differ from other
prokaryotic organisms in their mycelial life cycle and in possessing a
large (8-megabase pair), linear and GC-rich (about 72%) chromosome (10, 11). Recent discoveries suggest that replication of the linear
chromosome of Streptomyces coelicolor A3(2) proceeds
bidirectionally from the centrally located oriC region
toward the ends of the chromosome (12).
The key elements of initiation of the Streptomyces
chromosome replication, oriC region and DnaA protein, show
higher complexity than those of E. coli. The
Streptomyces oriC region contains numerous DnaA boxes, which
are grouped into two clusters (13, 14). The Streptomyces
DnaA protein consists, like all other DnaA proteins, of four domains.
In contrast to the other bacteria, the Streptomyces DnaA
protein is larger (70-73 kDa), since it comprises an additional stretch (~230 amino acids) of predominantly acidic or hydrophobic amino acids within domain II. The residues lower the isoelectric point
of the entire Streptomyces DnaA protein (pI = 5.7) (15, 16). As it was shown for the E. coli and Bacillus
subtilis DnaA proteins, domain III and the C-terminal part (domain
IV) of the Streptomyces DnaA protein are responsible for
binding of ATP and DNA, respectively (15, 17). The consensus sequence
of the Streptomyces DnaA box in oriC is
5'-TTGTCCACA-3', which differs at the third position (A'G) from the
E. coli DnaA box (13). In contrast to E. coli,
the Streptomyces DnaA protein can form a dimer when binding
to a single DnaA box. Recently, it has been shown that the domains I
and III are independently involved in dimerization of the
Streptomyces lividans DnaA protein molecules. The
interaction of Streptomyces DnaA protein with two DnaA boxes is cooperative and accompanied by strong DNA bending (16, 18). However,
we do not know the contribution of the different domains of the
Streptomyces DnaA protein to cooperative binding.
In this work, we apply a combined
PCR-EMSA1 technique (19) to
elucidate DNA sequence requirements for Streptomyces DnaA
protein binding. Using EMSA, we define details of the binding of the
Streptomyces wild type DnaA and its truncated forms to DNA.
We reveal that the domain I participates in cooperative DnaA
protein-DNA interactions. Quantitative analysis of gel mobility shifts
allowed us to determine binding constants for dimerization and
cooperative DNA protein interactions. In addition, we have varied the
spacing between two DnaA boxes and examined the consequences on
dimerization and cooperative binding of DnaA protein to these boxes.
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EXPERIMENTAL PROCEDURES |
Proteins and DNA--
The His-tagged wild type DnaA proteins of
S. lividans and its truncated mutants DnaA(III-IV)
comprising the domain III and the DNA binding domain were purified on a
Ni2+-nitrilotriacetic acid-agarose column (Qiagen) as
described earlier (15). The DNA binding domain of the DnaA protein
DnaA(BD) was expressed as a C-terminal glutathione
S-transferase fusion and purified using
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) followed by
factor Xa cleavage as described earlier (16).
Oligonucleotides were chemically synthesized and purified by high
performance liquid chromatography. For the kinetic analysis of DnaA
protein binding complementary nucleotides were annealed by heating at
95 °C for 10 min and gradually cooling to room temperature. The
oligonucleotides were end-labeled with [
-32P]ATP and
T4 polynucleotide kinase and purified from 5% nondenaturing polyacrylamide gels.
In Vitro DNA Binding Site Selection--
The recognition motif
of the DnaA protein was determined using several cycles of
amplification and selection, essentially as described previously (19).
The library of 58-bp oligonucleotides for the first selection cycle
was prepared by PCR using template oligonucleotides:
5'-GGCGGATCCTCGACTAGCGN20GCTACGAGCTGAGCTCGCG-3' and the primer pair 5'-GGCGGATCCTCGACTAGCG-3' and
5'-CGCGAGTCTAGCTCGTAGC-3' (restriction sites
BamHI and SacI are in italic type). The
amplification reaction was carried out in 100 µl using 1 pmol of
template oligonucleotide (1 pmol corresponds to the number of all
possible combinations of 20 degenerated bases) and 100 pmol of each
primer for 20 cycles, with each cycle consisting of 15 s at
96 °C, 30 s at 60 °C, and 30 s at 72 °C.
Double-stranded oligonucleotides were separated on 3% agarose gels,
electroeluted for 1 h to TBE buffer, and purified by
phenol-chloroform extraction and glycogen-ethanol precipitation. The
oligonucleotides were 5'-end-labeled with [
-32P]ATP
and T4 polynucleotide kinase and then incubated with DnaA protein in
concentrations of 5 or 50 nM in binding buffer (20 mM HEPES-KOH, pH 8.0, 5 mM magnesium acetate, 1 mM Na2EDTA, 4 mM dithiothreitol,
0.2% Triton X-100, 5 mg/ml bovine serum albumin, and 100 µM ATP) The reaction was carried out in 20 µl for 30 min at room temperature in the presence of competitor, poly(dI-dC), at
a concentration of 2.5 µg/µl. The complexes were separated on 5%
nondenaturing polyacrylamide gels in 0.25× TBE. The bands corresponding to DNA-protein complexes were excised, and the DNA was
eluted into gel elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2) for 3 h
at 60 °C and recovered by phenol-chloroform extraction followed by
glycogen-ethanol precipitation. The DNA was then amplified by PCR at
conditions described above and subjected to five additional selection
cycles of binding and amplification. The subsequent cycles differed
only in the amount of competitor added to binding reactions (for cycles
2-6, we used 5, 7.5, 20, 50, and 100 µg/µl, respectively).
Selected oligonucleotides after the fourth, fifth, and sixth round were
digested with BamHI and SacI and cloned into a
pUC19 vector. The DNA from independent clones was recovered using a
Spin Miniprep Kit (Qiagen) and subjected to sequencing using
32P-5'-end-labeled "
47" primer (New England Biolabs)
and the Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech).
EMSA and Determination of Equilibrium Constants--
For binding
assays, 5'-32P-end-labeled DNA (Table
I;
0.01 nM) was incubated
with different amounts of the DnaA proteins (concentration range
indicated in the legend to Fig. 2) in the presence of a competitor
(poly(dI-dC), 10 ng/µl) at 20 °C for 30 min in binding buffer (20 mM HEPES-KOH, pH 8.0, 5 mM magnesium acetate, 1 mM Na2EDTA, 4 mM dithiothreitol,
0.2% Triton X-100, 5 mg/ml bovine serum albumin, and 100 µM ATP). The free DNA and complexes were separated by
electrophoresis on 5% native polyacrylamide gels, prerun for 1 h
(0.25× TBE, 6 V/cm, 20 °C). Following electrophoresis, the
radioactive gel was dried and analyzed using a PhosphorImager and
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). The
variation in experimental data was evaluated by repeating each
experiment three or four times. Different protein titration experiments
showed variations up to 15%. Equilibrium constants were determined
using the modified statistical mechanical model (20) supplemented with
the protein dimerization module. For the DNA substrate containing a
single binding site A, the concentration of each species in the gel was
expressed by the following equations (model 1),
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Table I
Oligonucleotides used in the gel retardation experiments
The black arrow shows the strong Streptomyces DnaA box; the
white arrow shows the E. coli DnaA box R1/R4; the grey arrow
shows the box2 found by the in vitro DNA binding sites
selection assay; and the crossed out arrow shows the scrambled DnaA
box.
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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where S, SL, and
SL2 represent concentrations of free DNA,
monomer, and dimer respectively; L is protein (ligand)
concentration; kA is the microscopic equilibrium
affinity constant for binding site A, and kDi is the
affinity constant describing the dimerization process.
For the DNA containing binding sites A and B, the concentrations of the
species presented in the gel are described by the following equations
(model 2),
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(Eq. 5)
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(Eq. 6)
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(Eq. 7)
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(Eq. 8)
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where kB is the intrinsic affinity constant
for the binding site B, and kAB is the cooperativity
constant describing interaction of protein molecules occupying both
binding sites.
Experimental data were simultaneously fitted to the equations using
Scientist® for WindowsTM software (MicroMath).
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RESULTS |
Determination of the Optimal DnaA Protein Binding Site--
The
consensus sequence of the DnaA box identified within the
Streptomyces oriC region is 5'-TTGTCCACA-3'. The
Streptomyces oriC region contains a higher number of the
DnaA boxes (19 boxes) than the E. coli oriC region (five
boxes). A DNase I footprint of the oriC region with DnaA
showed protection of all boxes. However, when analyzed individually by
surface plasmon resonance (SPR), only some DnaA boxes were specifically
recognized by the DnaA protein (14, 15). To characterize in detail the
recognition properties of the Streptomyces DnaA protein, we
applied a binding site selection technique based on the combined EMSA
and polymerase chain reactions (Fig.
1A; Ref. 19).

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Fig. 1.
In vitro DNA binding sites
selection assay. A, the experimental design of the
test. B, DNA substrate and the primer pair used in the
assay. N, any nucleotide; boldface
letters, restriction sites BamHI and
SacI. C, subsequent rounds of the DnaA binding
site determination. Lanes 1, 2, and
3 in each cycle correspond to 0, 5, and 50 nM
concentration of the wild-type DnaA protein, respectively.
Frames indicate gel fragments from which DNA used in the
next round and/or sequencing was extracted. * and ** signify DNA bands
from which selection toward monomer and dimer started.
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The purified DnaA protein was incubated with a substrate pool of
double-stranded oligonucleotides, in which a random 20-bp region was
flanked by defined 19-bp sequences containing restriction sites to
facilitate subsequent cloning. The protein-DNA complexes were separated
from the unbound DNA on a 5% polyacrylamide nondenaturing gel. Only
one protein-bound fraction was visible. The DNA recovered from the
complex was amplified by PCR with the pair of primers complementary to
the defined sequences of the oligonucleotides (Fig. 1B) and
used as a substrate for the renewed binding assay. In the third
selection cycle, we observed a second retarded band with a higher
electrophoretic mobility than the previous one. Here, the bands with
lower and higher electrophoretic mobility are called a "dimer"
(Fig. 1C, ** complex) and a "monomer" (Fig. 1C, * complex), respectively. The DNA from both
bands was recovered and used independently in the next selection
cycles. The alignments of the selected
oligonucleotides after the fourth, fifth, and sixth cycle are shown in
Tables II and
III.
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Table II
Selected DNA binding sites for DnaA ("dimer" band)
The sequence of cloned binding sites for DnaA, derived after the
fourth, fifth, and sixth selection cycles, were aligned for maximum
match to the canonical DnaA box definition. The region of 20 originally
random nucleotides is shown in uppercase letters, whereas nucleotides
from adjacent primer regions are indicated by lowercase letters (only
five bases from each side of the random region are shown). Letters in
boldface type match the DnaA box consensus sequence. Letters in italic
type indicate the lower strand of the DnaA box. For the consensus
sequence, letters in boldface represent nucleotides present more than
85%. Uppercase letters indicate nucleotides appearing in the frequency
range 50-85%. Nucleotides that are represented at less than 15% were
considered as not significant at the indicated position.
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Table III
Selected DNA binding sites for DnaA ("monomer" band)
The sequence of cloned binding sites for DnaA, derived after the
fourth, fifth, and sixth selection cycles, were aligned for maximum
match to the canonical DnaA box definition. The region of 20 originally
random nucleotides is shown in uppercase letters, whereas nucleotides
from adjacent primer regions are indicated by lowercase letters (only
five bases from each side of the random region are shown). Letters in
boldface type match the DnaA box consensus sequence. Letters in italic
type indicate the lower strand of the DnaA box. For the consensus
sequence, letters in boldface represent nucleotides present more than
85%. Uppercase letters indicate nucleotides appearing in the frequency
range 50-85%. Nucleotides that are represented at less than 15% were
considered as not significant at the indicated position.
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The sequences selected from the "dimer" band after four rounds
reveal remarkable features: 63 out of the 67 oligonucleotides analyzed
carry two DnaA boxes, one complete 9-bp DnaA box, the "box 1," and
one incomplete DnaA box, the "box 2" ("box" as defined by
sequence). The incomplete box 2 consists of 4-7 bp (3'-part of
the DnaA box). Both boxes face each other and are adjacent (except for
a few oligonucleotides). 80% of the boxes 1 exhibit the E. coli type of DnaA box (T or A at the third position), and only
20% of them exhibit the Streptomyces type (G at the third position). During the next two rounds, the selection progressively narrowed the spectrum of observed oligonucleotide sequences; after six
rounds the box 1 is exclusively represented by the E. coli DnaA box R1/R4 (5'-TTATCCACA-3'), and the partial box 2 contains in
nearly all cases the essential T nucleotides at the 7'- and 9'-positions.
More than half of the oligonucleotides selected from the monomer band
after four rounds contained also two boxes. However, the monomer band
had only appeared after the third round of the selection (Fig.
1C), and therefore it still contained traces of the
oligonucleotides from the dimer band that were subsequently amplified.
After the next two selection rounds, only one out of the 44 oligonucleotides contained two boxes. All of the boxes 1 from the
monomer band exhibit the E. coli type DnaA box (R1/R4).
The data show that under the conditions of the assay, the
Streptomyces DnaA protein interacts only with DnaA boxes; no
other consensus sequences (e.g. the newly identified 6-bp
ATP-DnaA box (9)) have been found. The results, presented in Tables II
and III, show that DnaA protein from Streptomyces possesses
a higher affinity toward DnaA boxes from E. coli than toward
those from its own oriC region. Binding of the DnaA protein
as a dimer apparently requires "head-to-head" orientation of the boxes.
Kinetic Studies with Streptomyces DnaA Protein--
Upstream of
the promoter region of the Streptomyces dnaA gene are two
closely spaced DnaA boxes: a strong one (with the preferred Streptomyces sequence: 5'-TTGTCCACA-3') and a weak one
(5'-TTGTCCCCA-3') in head-to-head arrangement with 3 bp in between
(21). Interaction of the DnaA protein with these boxes creates an
autoregulatory circuit similar to that known for the E. coli
dnaA gene (21). Recently, we have shown that binding of the DnaA
protein to both DnaA boxes exhibits a cooperative character (16).
Domains I and III independently participate in the dimerization of the
DnaA protein molecules (18). To evaluate in detail the kinetics of binding of the DnaA protein to DnaA boxes from the dnaA gene
promoter region, we applied gel mobility shift assays to determine the binding constants for cooperativity as well as for dimerization (20).
This assay permits the quantitative analysis of the individual protein-DNA complexes. In our gel retardation experiments, we used the
wild-type DnaA protein and its truncated forms, the DnaA(III-IV) lacking the two N-terminal domains (I and II) and the DnaA(BD) containing only the DNA binding domain IV. As a prerequisite for the
kinetic studies, two DNA substrates containing either a single strong
DnaA box, 1s-0 (the weak DnaA box was scrambled), or
two DnaA boxes, 2s, derived from the promoter
region of the dnaA gene were designed in such a way that the
DnaA box(es) is flanked on both sides by sequences corresponding to
those within the dnaA promoter region (Table I). To simplify
kinetic calculations, the 2s substrate contains two
identical boxes; the weak DnaA box was replaced by the strong one
(Table I). For the analysis, we used a fixed input DNA concentration
and various protein concentrations, spanning 4 orders of magnitude (see
legend to Fig. 2). The concentration of
DNA in the reaction mixture was chosen to be at least 5 times lower
than the lowest protein concentration used.

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Fig. 2.
Binding of the DnaA protein forms to
Streptomyces DnaA boxes. A, gel
retardation experiments with three DnaA protein forms and DNA
substrates containing one or two Streptomyces DnaA boxes.
The binding to oligonucleotides 1s-0 and 2s of
the wild-type protein (a and b), DnaA(III-IV)
(c and d), and DnaA(BD) (e and
f) are shown. In each panel, protein
concentrations are rising from left to right. For
DnaA wild type (wt) (a and b),
lanes 1-18 indicate 0, 0.1, 0.18, 0.32, 0.57, 1.0, 1.8, 3.2 ... 1000 nM, respectively; for
DnaA(III-IV) and DnaA(BD) (c-f), lanes
1-18 indicate 0, 0.18, 0.32, 0.57, 1.0, 1.8, 3.2, 5.7 ... 1800 nM. B, quantitative analysis
of the DnaA wild type-2s interaction. Intensity
(concentration) of every band from the gel presented in A
(b) was expressed as a ratio of total lane intensities; band
intensity was measured for the area (square) containing a given band
and the area separating it from the band with higher mobility.
Rhombuses indicate concentrations of free DNA;
squares and triangles indicate the concentrations
of monomer and dimer bands, respectively. Using the Scientist® for
WindowsTM program and kA and
kDi constants found in band shift assays with a
single DnaA box (a, c, and e), the
points on the plot were simultaneously fitted to functions describing
the concentrations of band species (model 2). Dashed
lines present best fitted curves for band concentration
functions.
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According to the statistical mechanical approach (20), the interaction
between the DnaA protein and the DNA substrate composed of two binding
sites could be described by the following equations,
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(Eq. 9)
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(Eq. 10)
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(Eq. 11)
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(Eq. 12)
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where S, SL, and SL2
are concentrations of free DNA, monomer, and dimer bands, respectively;
L is protein (ligand) concentration; kA
and kB are the microscopic equilibrium affinity constants for binding sites A and B, respectively; and
kAB is the cooperativity constant describing the
interaction of protein molecules occupying both binding sites.
These equations contain parameters kA,
kB, and kAB that occur only in
combination and therefore cannot be unambiguously determined. However,
if one of the pair kA or kB is
known, it is possible to determine the remaining two. One way to
achieve that goal is carrying out the gel shift experiment using a
substrate containing only one binding site. In our analysis, it is the
1s-0 oligonucleotide. Concentration of free DNA or
DNA-protein complexes in this system is given by the Langmuir
isotherm.
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(Eq. 13)
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The interaction of the 1s-0 DNA fragment with
increasing amounts of the wild type DnaA and DnaA(III-IV) led to two
complexes (Fig. 2A, a and c),
whereas incubation with the DnaA(BD) resulted in a single complex even
at the highest protein concentration (Fig. 2A,
e). Formation of a dimer band by the first two DnaA derivatives could be explained by an unspecific interaction with the
scrambled DnaA box or another sequence motif of the 1s-0
oligonucleotide or a protein dimerization process occurring on the DNA
with only one-half of the dimer bound to DNA. The first possibility can be excluded, because domain IV alone does not form slower migrating complexes on that substrate, and all three proteins do not interact with DNA in which both DnaA boxes are scrambled (data not shown). Taking into account the dimerization process, we modified the original
equations by adding a constant describing the oligomerization reaction.
As a result, we obtained two sets of equations (model 1 and model 2)
for the interaction with a single (1s-0) and with two DnaA
boxes (2s), respectively (see "Experimental
Procedures").
The affinity constant for the single DnaA box, kA,
and the affinity constant describing the dimerization process, kDi, were calculated by analyzing the interaction of the wild-type DnaA (or its truncated forms) with the 1s-0
substrate according to model 1 or its simplified version, the Langmuir
isotherm for the DnaA(BD). The values obtained from those experiments
allowed us to determine kB and
kAB, the intrinsic affinity constant for the second
DnaA box in substrate 2s, and the cooperativity constant,
respectively (Fig. 2B).
The wild type DnaA protein and its truncated forms exhibit similar
affinity to the single DnaA box, the 1s-0 substrate (Table IV; Kd = 4-10
nM). The affinity constants calculated for the second DnaA
box from the 2s substrate (kB) were found
to be nearly identical to the kA values. Thus, the
affinities seem to be independent of flanking sequences of the DnaA
box(es).
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Table IV
Characterization of DNA binding properties of three DnaA protein
forms to DnaA boxes derived from the promoter region of the dnaA
gene
For every constant determination, S.D. is given (expressed as a
percentage). *, corresponding dissociation constants
KD (reciprocal values of the affinity
constants).
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The calculated cooperativity constants, kAB, showed
that only the wild type DnaA is able to interact cooperatively with the
two DnaA boxes; the cooperativity effect increases the affinity of the
DnaA protein toward the DnaA boxes over 40 times. The values obtained
for DnaA(III-IV) and DnaA(BD) are very close to 1; therefore, we
consider these interactions as noncooperative. The results demonstrate
that domain I or II or both of them are responsible for the
cooperativity effect.
The dimerization dissociation constant for the wild-type protein (35 nM) is about 3 times lower than for a protein lacking domains I and II, DnaA(III-IV) (114 nM).
The Distance Separating DnaA Boxes Does Not Influence the
Cooperativity of the DnaA-DnaA Box Interaction--
The arrangement of
the 19 DnaA boxes occurring within the Streptomyces oriC
regions is highly conserved. The spacing between DnaA boxes varies from
3 to 20 bp or even more (except for two DnaA boxes that are adjacent).
To evaluate the influence of the distance that separates binding sites
on the cooperativity, we have designed three double-stranded
oligonucleotides with two strong DnaA boxes separated by various
spacings. The first one is the previously analyzed 2s
substrate that contains 3 bp between the two DnaA boxes. The
2s mimics the spacing between the fifth and sixth DnaA boxes
within the Streptomyces oriC region. The other two, 2s
+5 and 2s +10 (Table I), contain 5 and 10 additional base pairs between the DnaA boxes, respectively. In the 2s
+10 substrate, the increase of the distance separating
both boxes by 10 bp does not affect their positioning on the DNA helix, while in the 2s +5 variant, the two DnaA boxes lie on the
opposite face of the helix. The calculated cooperativity constants for the three substrates are presented in Table
V.
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Table V
Comparison of the cooperativity constants for three DNA substrates that
differ in the length of the spacer separating DnaA boxes
For every constant determination, S.D. is given (expressed as a
percentage).
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Adding a turn of the helix (2s +10) increased the
cooperativity about 2.5 times. Surprisingly, the addition of 5 bp (a
half helix turn) caused negligible change of the cooperativity. As it
was shown for the "wild-type" substrate, 2s, the
interaction of the truncated forms of the DnaA protein, DnaA(III-IV)
and DnaA(BD), with the 2s +5 and 2s +10
substrates exhibited a noncooperative character (Table V).
Streptomyces DnaA Protein Prefers DnaA Boxes of the E. coli
Type--
The binding selection assay suggested that the
Streptomyces DnaA protein prefers the DnaA box of E. coli type (5'-TTATCCACA-3') over its own DnaA box
(5'-TTGTCCACA-3'). To evaluate in detail the interaction of the DnaA
protein with the E. coli type DnaA box, quantitative gel
retardation assays were performed. As before, we used a two DnaA box
system that enabled us to evaluate cooperativity in addition to
intrinsic affinity constants. Thus, two substrates were used for the
calculations: Sel 1s-0 and Sel 2s, containing the
single box 1 (E. coli DnaA box) or two adjacent boxes 1 in head-to-head orientation, respectively (Table I). The Sel
1s-0 substrate was used to determine the affinity constant for the single binding box and the dimerization affinity constant. These values
were then used for the interaction analysis of the DnaA protein with
two boxes (substrate Sel 2s). Since the wild-type DnaA
protein formed dimers during the interaction with a single DnaA box,
the same mathematical models (models 1 and 2; see "Experimental Procedures") were applied. In addition to the substrate with two identical boxes, the Sel 1s-1/2 oligonucleotide selected
after the sixth cycle of the binding assay was also analyzed. The
results of this analysis and their comparison with the constants
obtained for the wild-type DnaA boxes from the promoter region are
summarized in Table VI.
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Table VI
Comparison of the binding properties of wild type DnaA protein to the
DnaA boxes derived from the promoter region of the dnaA gene and
oligonucleotides obtained in the optimal site selection
For every constant determination, S.D. is given (expressed as a
percentage). *, corresponding dissociation constants
(reciprocal values of the affinity constants). **, this
substrate comprised only one DnaA box; kB does not
exist. ***, the intrinsic affinity constants for the binding
sites A and B were found to be not identical. White and black arrows
indicate E. coli- and Streptomyces-like DnaA
boxes, respectively. The gray arrow indicates box 2 found by the
in vitro DNA binding site selection
assay.
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The affinity of the wild-type DnaA to the single DnaA box of E. coli is approximately 4 times higher than its affinity to the
strong Streptomyces DnaA box. It explains why such DnaA
recognition sequences had been found in the selection binding assay. As
expected, the dimerization constant does not depend on the DNA
recognition sequence; its value is nearly identical for both types of
DnaA boxes (Table VI). The cooperativity constant for the Sel
2s substrate is approximately 2 times lower than the cooperativity
for the wild type Streptomyces DnaA boxes (2s
substrate). This may be due to the fact that the selected sequences did
not have the 3-bp spacer.
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DISCUSSION |
Despite extensive work on the mechanism of initiation of DNA
replication in prokaryotic and eukaryotic systems, several critical aspects of this mechanism and its control still remain obscure. One
significant gap is a lack of understanding of the biological and
biochemical roles of multiple initiator protein binding sites required
by a large group of plasmid (interons) and chromosomal replicons (DnaA
boxes). The oriC region of the Streptomyces
linear chromosome consists of 19 DnaA boxes that serve as binding
sites. Therefore, Streptomyces provides a good model for
studying the interaction of initiator protein with multiple cognate
binding sites. The initiator protein DnaA of Streptomyces
contains two dimerization domains (I and III) that are separated by a
long flexible domain II. According to our preliminary results (16, 22),
interaction of the DnaA protein with two DnaA boxes exhibits cooperativity (e.g. the DnaA protein specifically binds to
DNA fragments with two "weak" DnaA boxes, which are not bound as
individual boxes). Here, we present the recognition properties of the
Streptomyces DnaA protein and the kinetic details of
dimerization and cooperative DNA binding of this protein. Finally, we
try to answer the questions of why the Streptomyces oriC
region contains so many DnaA boxes and how it arose.
The Streptomyces DnaA Protein Prefers the E. coli DnaA Box over Its
Own DnaA Box--
Analysis of 57 DnaA boxes from three different
Streptomyces oriC regions showed that the preferred sequence
is 5'-TTGTCCACA-3' ("strong" DnaA box). However, each of the
analyzed oriC regions contains only one strong DnaA box,
while the sequences of others, the weak DnaA boxes, differ by one or
two bases from the preferred sequence. Our previous results revealed
that the Streptomyces initiator protein DnaA interacts
specifically only with a few isolated (14) boxes, including the strong
one from its own oriC region (Table
VII). To evaluate the recognition
properties of the Streptomyces DnaA protein, a binding
selection assay based on a combined PCR-EMSA technique was applied.
Unexpectedly, we found that the Streptomyces DnaA protein
prefers the E. coli DnaA box instead of its own DnaA box.
After four cycles of binding, only a fraction of the selected
oligonucleotides contained Streptomyces-like DnaA boxes,
whereas after subsequent cycles (fifth and sixth), this type of DnaA
box did dot appear at all. The results are also supported by the gel
retardation experiments (Table VII). The Streptomyces DnaA
protein shows about 4 times higher affinity for the E. coli DnaA box (Kd = 2.6 nM) than for its own
strong DnaA box (Kd = 10.4 nM);
therefore, the E. coli box was exclusively selected during
the PCR-EMSA binding assay. The data obtained by gel retardation are
consistent with the dissociation constants determined by surface
plasmon resonance; the affinity of the Streptomyces DnaA
protein to the E. coli DnaA box and to the strong
Streptomyces DnaA box have been calculated to be
Kd = 3.4 nM and Kd = 12 nM, respectively (Table VII) (16, 17).
The nucleotides immediately adjacent to the selected binding box
exhibit low diversity (5'-(A/g)(G/C)-box 1-(G/a)-3'); therefore, the possibility cannot be excluded that these sequences influence the
binding affinity. The naturally occurring DnaA boxes are usually flanked by G from both sides (5'- (a/c/g/t)(G/c)t-DnaA-G-3').
Cooperativity and Dimerization: Features of the Streptomyces DnaA
Protein--
So far, mainly cooperative DNA binding of transcription
factors has been studied extensively (23). Cooperativity is best understood for
cI repressor. The recently obtained crystal
structure of the cI repressor C-terminal domain provided a
comprehensive study of the molecular basis of cooperativity (24). In
eukaryotes, activation of genes frequently requires the cooperative
assembly of large protein complexes on the DNA. However, cooperative
binding may also serve functions other than regulation of gene
expression. Protein-protein interactions that result in cooperative DNA
binding may lead to bending and/or looping of the intervening DNA,
contributing to the formation of higher order structured DNA-protein
complexes. According to our previous studies, the formation of the
Streptomyces initial nucleoprotein complex involves the
sequential binding of the DnaA protein molecules to 19 DnaA boxes.
Subsequent protein-protein interaction leads to bending and looping of
the Streptomyces oriC region (18).
Our recent experiments indicate that the wild-type
Streptomyces DnaA protein binds two or more DnaA boxes in a
cooperative manner (16). E. coli DnaA protein also binds
cooperatively to two nonamer DnaA boxes and to three hexamer ATP-DnaA
boxes located within the dnaA promoter region (9). Here, we
have shown that the truncated DnaA protein lacking domains I and II is
deficient in cooperative binding to two adjacent sites. Thus, domain I
(or domains I and II) is responsible for this effect. However, the domain II of the DnaA proteins is highly variable and does not contain
any relevant secondary structure elements. Therefore, it is not a
plausible candidate for any functional role.
The wild-type DnaA protein binds to two DnaA boxes separated
by various spacings with a cooperativity parameter ranging from 23 to
over 100. Surprisingly, cooperativity does not depend severely on the
spacer length separating both binding sites (Table V). Adding a turn of
the helix (2s +10) increased the cooperativity about 2.5 times, whereas the addition of 5 bp (a half of a helix turn) had a
modest effect on the cooperativity. Removing of 3 bp (Sel
2s) resulted in only 50% reduction of the cooperativity parameter. However, the Sel 2s oligonucleotide consists of
the E. coli type DnaA boxes and therefore cannot be directly
compared with other DNA fragments. The moderate influence of the spacer length on the cooperativity may suggest that the protein domains involved in the intramolecular reactions are very flexible. Therefore, changing the spacing between adjacent binding sites does not affect the
ability of the DnaA protein to bind in a cooperative manner. The
spacing between DnaA boxes within the oriC region varies
from 3 to 20 bp. Thus, in theory, the DnaA protein could be able to interact cooperatively with each pair of the adjacent DnaA boxes. However, in the binding site selection assay, only DnaA boxes that face
each other have been selected. Therefore, we speculate that
cooperativity occurs only when the binding sites are oriented head-to-head (such a box arrangement has been found in the promoter region of the dnaA gene as well as in the oriC
regions, e.g. the fifth and sixth DnaA boxes). Our DNase I
footprinting experiments (17) and electron microscopy studies (18)
corroborate this hypothesis. DNase I footprinting experiments showed
that in the oriC region, at the low protein concentration,
DnaA binds first to the fifth and sixth DnaA boxes. According to the
electron microscopy studies, the highest incidence of protein binding
occurred at the middle of the first cluster of DnaA boxes, which
corresponds to the location of DnaA boxes 5 and 6. Therefore, we assume
that cooperativity at close distance determines at least the start of
DnaA-oriC complex formation. Additional long range
interactions may be formed subsequently and may be responsible for loop formation.
We established the kinetic constants for dimerization of the DnaA
protein. The Streptomyces DnaA is the first chromosomal initiator for which the kinetics of two dimerization domains, I and
III, have been determined. These domains dimerize independently (18).
The dimerization of the DnaA protein does not occur in the absence of
DNA. The wild-type DnaA protein reveals 3 times higher dimerization
capability than the truncated DnaA protein containing only one
dimerization domain.
The intermolecular interactions of the DnaA protein are 3-20 times
weaker than the interactions between DnaA protein and its DNA target
(Table IV). Probably, it facilitates effective interactions of the DnaA
molecules with DNA containing multiple recognition sequences
(e.g. within the oriC region) and further
formation of the nucleoprotein complex. For E. coli DnaA,
the N-terminal domain 1 has been shown to promote oligomerization (25).
However, as for Streptomyces DnaA, a second interaction face
has been postulated in domain 3 or 4 (26).
Why Does the Streptomyces oriC Region Contain so Many DnaA
Boxes?--
The GC content of 57 Streptomyces DnaA boxes
derived from three Streptomyces oriC regions is about 10 and
20% lower than the GC content of the oriC region (63%) and
the overall GC content of S. lividans DNA (72%),
respectively. However, it is still significantly higher than the GC
content of the average E. coli DnaA box (~30%). This
difference in GC content explains the difference in the DnaA box
consensus sequence between Streptomyces (5'-TTGTCCACA-3') and E. coli (5'-TTATCCACA-3'). It also results in
flanking sequences of Streptomyces DnaA boxes that are
relatively rich in GC.
As shown in Table VII, the dissociation constant
(Kd) for specific binding of individual DnaA boxes
derived from the S. lividans oriC region varies between 10 and 78 nM; a few additional DnaA boxes are not recognized
by DnaA if they are analyzed outside the context of oriC
(Kd exceeding 200 nM). Interestingly, the affinity of the S. lividans DnaA protein for the R1/R4
E. coli DnaA box (Kd = 3.4 nM) is ~3 times higher than its affinity for the strong
Streptomyces DnaA box. The apparent dissociation constant
for binding of the E. coli DnaA protein to the DnaA box R1/R4 was calculated to be Kd = 1.1 nM
(7).
Despite the strong difference in GC content between
Streptomyces and other microoganisms, including E. coli, the domains I and III and the binding domain of the
Streptomyces DnaA are highly conserved. A consequence of the
high GC content of Streptomyces structural genes (72-74%)
is the very nonrandom codon usage, with an extreme paucity of codons
with A or T in the third position (C or G is usually at the third codon
position). Like E. coli DnaA protein, the binding domain of
Streptomyces DnaA protein contains the same putative DNA
binding motive: two amphipathic
-helices with the basic loop in
between followed by a third long
-helix (6, 16).
The affinity of the S. lividans protein for DNA fragments
containing two or three closely spaced DnaA boxes is 6-10 times higher
than its affinity for the single strong DnaA box (Table VII) and is
comparable with the affinity of the E. coli DnaA protein to
the R1/R4 box (0.9-1.2). The data suggest that efficient binding of
the Streptomyces DnaA protein to DNA requires the presence of more than one Streptomyces DnaA box. Therefore, due to
the high GC pressure exerted during the course of
Streptomyces evolution, the structure and the sequence of
oriC region has been changed.
The relatively low affinity of Streptomyces DnaA protein for
a single Streptomyces DnaA box seems to be compensated by a
high number of DnaA boxes that are bound in a cooperative manner. In comparison with the
cI repressor, the cooperativity parameter of the
wild-type DnaA protein () is moderate;
cI repressor by binding to its three operator sites shows cooperativity in the range of
250-950 (20). Such high values in a system with 19 binding sites
(Streptomyces oriC) might cause irreversible binding of DnaA
protein to the oriC region and consequently would block subsequent replication steps.