From the Department of Human Biological Chemistry and Genetics and
the Sealy Center for Structural Biology, University of Texas Medical
Branch at Galveston, Galveston, Texas 77555-1053
Fluorescence energy transfer experiments provide direct proof that the
DnaB hexamer binds ssDNA in a single orientation, with respect to the
polarity of the sugar-phosphate backbone. This is the first evidence of
directional binding to ssDNA of a hexameric helicase in solution. The
strong binding subsite is close to the small 12-kDa domains of the DnaB
hexamer and occludes the 5'-end of the ssDNA. The strict orientation of
the helicase on ssDNA indicates that, when the enzyme approaches the
replication fork, it faces double-stranded DNA with its weak subsite.
The data indicate that the different binding subsites are located
sequentially, with the weak binding subsite constituting the entry site
for double-stranded DNA of the replication fork.
 |
INTRODUCTION |
The DnaB protein is an essential replication protein in
Escherichia coli (1) which is involved in both the
initiation and elongation stages of DNA replication (2-4). The protein
is the E. coli primary replicative helicase, i.e.
the factor responsible for unwinding the duplex DNA in front of the
replication fork (5, 6). The DnaB protein is the only helicase required
to reconstitute DNA replication in vitro from the
chromosomal origin of replication. In the complex with
ssDNA,1 the DnaB protein
forms a "mobile replication promoter." This nucleoprotein complex
is specifically recognized by the primase in the initial stages of the
priming reaction (1).
In solution, the native DnaB protein exists as a stable hexamer,
composed of six identical subunits (7-9). Sedimentation equilibrium,
sedimentation velocity, and nucleotide cofactor binding studies show
that the DnaB helicase exists as a stable hexamer in a large protein
concentration range, specifically stabilized by magnesium cations (7,
8). Hydrodynamic and electron microscopy data indicate that six
protomers aggregate with cyclic symmetry in which the protomer-protomer
contacts are limited to only two neighboring subunits (7, 10, 11).
Sedimentation velocity and electron microscopy studies reveal that the
DnaB hexamer undergoes dramatic conformational changes upon binding
AMP-PNP and ssDNA, and provide direct evidence of the presence of long
range allosteric interactions in the hexamer, encompassing all six
subunits of the enzyme (8, 11).
Recently, we obtained the first estimate of the stoichiometry of the
DnaB helicase-ssDNA complex and the mechanism of the binding (12-14).
Using the quantitative fluorescence titration method, we determined
that the DnaB helicase binds ssDNA with a stoichiometry of 20 ± 3 nucleotides/DnaB hexamer and that this stoichiometry is independent of
the type of nucleic acid base (13). Our thermodynamic studies of
binding of ssDNA oligomers to the DnaB hexamer show that the enzyme has
a single, strong binding site for ssDNA (12). The results also show
that the same binding site is used in the binding to oligomers and
polymer nucleic acids (12, 13). Moreover, photo-cross-linking
experiments indicate that the ssDNA binding site is located
predominately, if not completely, on a single subunit of the hexamer
(12, 13).
The reaction catalyzed by a helicase, the unwinding of a duplex DNA,
must take place in the DNA binding site. The fact that the helicase
uses the same single DNA binding site, when forming a complex with
polymer ssDNAs, oligomers, and replication fork substrates, indicates a
complex structure of the nucleic acid binding site that can accommodate
both ssDNA and dsDNA.
In this communication, we report the analysis of interactions between
the DnaB helicase and DNA within the total DNA binding site of the
enzyme. We present direct evidence that the total DNA binding site of
the helicase is structurally and functionally heterogeneous. The total
binding site is built of two subsites, each encompassing approximately
10 nucleotide residues. We provide direct proof that the DnaB hexamer
binds ssDNA in a strictly single orientation, with respect to the
polarity of the sugar-phosphate backbone of the nucleic acid. The
results indicate that the binding subsites are sequentially located
along the nucleic acid lattice, with the weak binding subsite
constituting an entry site for the duplex part of the replication
fork.
 |
MATERIALS AND METHODS |
Reagents and Buffers--
All solutions were made with distilled
and deionized >18 megaohms (Milli-Q Plus) water. All chemicals were
reagent grade. Buffer T2 is 50 mM Tris adjusted to pH 8.1 with HCl, 5 mM MgCl2, 10% glycerol. Buffer H
is 50 mM Hepes adjusted to pH 8.1 with HCl, 5 mM MgCl2, 10% glycerol. The temperature,
AMP-PNP, and salt concentrations are indicated in the text. The
fluorescent markers, CPM, and fluorescein 5'-isothiocyanate, used in
the modification, were purchased from Molecular Probes (Eugene,
OR).
DnaB Protein--
The E. coli DnaB protein was
purified, as described previously by us (7, 15-17). The concentration
of the protein was spectrophotometrically determined, using extinction
coefficient
280 = 1.85 × 105
cm
1 M
1 (hexamer) (7).
Site-directed Mutagenesis of the DnaB Helicase--
Replacement
of the arginine residues at position 14 from the N terminus of the DnaB
protein and obtaining the DnaB protein variant, R14C, were performed
using the plasmid RLM1038, harboring the gene of the wild type DnaB
helicase, generously provided by Dr. R. McMacken. The site-directed
mutagenesis was accomplished in the NIEHS Center facility (National
Institutes of Health) directed by Dr. T. Wood.
Labeling the DnaB R14C Variant with Fluorescent
Markers--
Labeling of the 6 cysteine residues of the DnaB variant,
R14C hexamer, with CPM was performed in H buffer (pH 8.1, 100 mM NaCl, 5 mM MgCl2, 10% glycerol)
at 4 °C. The fluorescent label was added from the stock solution to
the molar ratio of the CPM/R14C ~25. The mixture was incubated for
4 h, with gentle mixing. After incubation, the protein was
precipitated with ammonium sulfate and dialyzed overnight against
buffer T2. Any remaining free dye was removed from the modified
R14C-CPM by applying the sample on a DEAE-cellulose column and eluting
with buffer T2 containing 500 mM NaCl. The degree of
labeling was determined by absorbance of the marker at 394 nm using the
extinction coefficient of CPM,
394 = 27 × 103 cm
1 M
1,
providing the value of 5.8 ± 0.1 of CPM per DnaB
hexamer.2
Nucleic Acids--
All nucleic acids were purchased from Midland
Certified Reagents (Midland, TX). The etheno-derivatives of nucleic
acids were obtained by modification with chloroacetaldehyde (12, 18). Oligomer dT(pT)19, labeled at the 5'-end with fluorescein,
5'-Fl-dT(pT)19, was synthesized using fluorescein
phosphoramidate (Glen Research). Labeling of the 3'-end was performed
by synthesizing dT(pT)19 with the last residue at the
3'-end of the oligomer having the amino group on a six-carbon linker.
The amino group was subsequently modified with fluorescein
5'-isothiocyanate to obtain dT(pT)19-Fl-3'. The degree of
labeling was determined by absorbance at 494 nm (pH 9), using the
extinction coefficient, 7.6 × 104
M
1 cm
1 (13). The same
procedures were used for labeling the 5'- and 3'-ends of the
dA(pA)9. The concentrations of labeled oligomers were
spectrophotometrically determined at 260 nm (pH 8.1), using extinction
coefficients, 1.76 × 105 M
1
cm
1 and 11.4 × 105
M
1 cm
1, respectively (13). The
concentrations of d
A(p
A)9, d
A(p
A)8, d
A(p
A)7, d
A(p
A)6,
d
A(p
A)5, d
A(p
A)4, and
d
A(p
A)3 were determined using extinction
coefficients 37 × 103, 33.3 × 103,
29.6 × 103, 25.9 × 103, 22.2 × 103, 18.5 × 103, and 14.8 × 103 M
1 cm
1 at 257 nm, respectively (12, 13, 19). Labeling the 5'-ends of ssDNA oligomers
with 32P was performed using the standard procedure
(12).
Sedimentation Velocity Measurements--
Analytical
sedimentation experiments were performed using an Optima XL-A
analytical ultracentrifuge. Analyses of the sedimentation runs were
performed as we previously described (8, 9, 13). The reported values of
sedimentation coefficients were corrected to standard conditions,
s20,w, for solvent density and viscosity
(7).
Fluorescence Measurements--
All steady-state fluorescence
measurements were performed using the SLM-Aminco 48000S and 8100 spectrofluorometers (20). The emission spectra were corrected for a
wavelength dependence of the instrument response using a software
provided by the manufacturer. The binding of the DnaB protein was
followed by monitoring the fluorescence of the etheno-derivatives of
ssDNA oligomers (
ex = 325 nm,
em = 410 nm). All titration points were corrected for dilution and, if
necessary, for inner filter effect using the formula (15),
|
(Eq. 1)
|
where Ficor is the
corrected value of the fluorescence intensity at a given point of
titration i, Fi is the experimentally
measured fluorescence intensity, Bi is the
background, Vi is the volume of the sample at a
given titration point, Vo is the initial volume of the sample, b is the total length of the optical path in the
cuvette expressed in centimeters, and
Ai
ex is the absorbance of the sample at the excitation wavelength. Computer fits were performed using KaleidaGraph software (Synergy Software, PA) and Mathematica (Wolfram Research, IL). The relative fluorescence increase
of the nucleic acid,
F, upon binding the DnaB protein is
defined by the equation,
|
(Eq. 2)
|
where Ficor is
defined by Equation 1, and Fo is the initial value
of the fluorescence of the same solution.
All steady-state fluorescence anisotropy measurements were performed in
the L format, using Glan-Thompson polarizers placed in the excitation
and emission channels. The fluorescence anisotropy, r, of
the sample was calculated by the equation,
|
(Eq. 3)
|
where I is the fluorescence intensity, and the first
and second subscripts refer to vertical (V) polarization of
the excitation and vertical (V) or horizontal (H)
polarization of the emitted light (16). The factor G = IHV/IHH corrects for the different sensitivity of the
emission monochromator for vertically and horizontally polarized light
(21). The limiting fluorescence anisotropies of fluorophores,
ro, were determined by measuring the anisotropy of a
given sample at different solution viscosity, adjusted by sucrose or
glycerol, and extrapolating to viscosity =
, using the Perrin
equation (22).
Determination of the Average Fluorescence Energy Transfer
Efficiency from CPM on the Small 12-kDa Domains of the DnaB Hexamer to
the Fluorescein Residue Attached at the 5'- or 3'-End of the ssDNA
Oligomers--
The efficiency of the fluorescence radiationless energy
transfer, E, from CPM (donor), located on the small 12-kDa
domains of the DnaB protein variant R14C, to the fluorescein
(acceptor), located at the 5'- or 3'-end of dT(pT)19, bound
in the DNA binding site of the helicase, has been determined using two
independent methods. The fluorescence of the donor in the presence of
the acceptor, FDA, is related to the fluorescence of
the same donor, FD, in the absence of the acceptor
by the equation,
|
(Eq. 4)
|
where
D is the fraction of donors in the complex with
the acceptor, and ED is the average fluorescence energy transfer from donor to acceptor, determined from the quenching of the donor fluorescence. Thus, the average transfer efficiency, ED, obtained from the quenching of the CPM
fluorescence upon binding of the labeled ssDNA oligomer, is obtained by
rearranging Equation 4,
|
(Eq. 5)
|
where, in the considered case, FD and
FDA are the values of the CPM fluorescence intensity
in the absence and presence of bound 5'-Fl-dT(pT)19 or
dT(pT)19-Fl-3'. The value of
D has been
determined using the binding constants of the 20- and 10-mers for the
DnaB helicase measured in the same solution conditions (13).
In the second independent method, the average fluorescence transfer
efficiency, EA, has been determined, using a sensitized acceptor fluorescence by measuring the fluorescence intensity of the acceptor (fluorescein) excited at 435 nm, where the
donor (CPM) predominantly absorbs, in the absence and presence of
R14C-CPM. The fluorescence intensities of the acceptor in the absence,
FA, and presence, FAD, of the
donor are defined as follows,
|
(Eq. 6)
|
and
|
(Eq. 7)
|
where Io is the intensity of incident light,
CAT and CDT are the total
concentrations of acceptor and donor,
A is the fraction of
acceptors in the complex with donors,
A and
D are
the molar absorption coefficients of acceptor and donor at the
excitation wavelength (435 nm), respectively;
FA and
BA are the quantum yields of
the free and bound acceptor; and EA is the average
transfer efficiency determined by acceptor-sensitized emission. All
quantities in Equations 6 and 7 can be experimentally determined. For
the case considered in this work, the acceptor is practically
completely saturated with the donor, i.e.
A = 1. Thus, for
A = 1, dividing Equation 7 by Equation 6 and
rearranging provides the average transfer efficiency as described by
the following.
|
(Eq. 8)
|
It should be pointed out that the energy transfer efficiencies,
ED and EA, are apparent
quantities. ED is a fraction of the photons absent
in the donor emission as a result of the presence of an acceptor,
including transfer to the acceptor and possible nondipolar quenching
processes induced by the presence of the acceptor, and
EA is a fraction of all photons absorbed by the
donor that were transferred to the acceptor. The true Förster
energy transfer efficiency, E, is a fraction of photons
absorbed by the donor and transferred to the acceptor in the absence of
any additional nondipolar quenching resulting from the presence of the
acceptor (22). The value of E is related to the apparent
quantities of ED and EA, by the
following (23).
|
(Eq. 9)
|
Thus, measurements of the transfer efficiency, using both
methods, are not alternatives but parts of the analysis used to obtain
the true efficiency of the fluorescence energy transfer process,
E.
The fluorescence energy transfer efficiency between donor and acceptor
dipoles is related to the distance, R, separating the dipoles by the equation,
|
(Eq. 10)
|
where Ro = 9790 (
2
n
4
d
J)
is the so called Förster critical
distance (in angstroms), the distance at which the transfer efficiency
is 50%;
2 is the orientation factor;
d is
the donor quantum yield in the absence of the acceptor; and
n is the refractive index of the medium (n = 1.4) (22). The overlap integral, J, characterizes the
resonance between the donor and acceptor dipoles.
The fluorescence transfer efficiency of chemically identical donor and
acceptor pairs, characterized by the same quantum yields, depends on
the distance between the donor and acceptor, R, and the
factor,
2, describing the mutual orientation of the
donor and acceptor dipoles (22). Although in the work presented in this
paper we are interested in relative distances between donors and
acceptors, evaluation of
2 allowed us to estimate the
effect of the orientation factor on the differences between the studied
donor-acceptor distances. The factor
2 cannot be
experimentally determined; however, the upper
(
2max) and lower
(
2min) limits of
2 can be
obtained from the measured limiting anisotropies of the donor and
acceptor and the calculated axial depolarization factors, using the
procedure described by Dale et al. (24). When both axial
depolarization factors are positive,
2max
and
2min can be calculated from
2max = (
)(1 + <dXD> + <dXA> + 3<dXD><dXA>)
and
2min = (
)(1
(1/2)(<dXD> + <dXA>), where
<dXD> and
<dXA> are the axial depolarization factors
for the donor and acceptor, respectively (24). The axial depolarization
factors have been calculated as square roots of the ratios of the
limiting anisotropies of the donors (CPM on the DnaB helicase) and
acceptors (fluorescein at the 5'- or 3'-end of the ssDNA oligomers) and their corresponding fundamental anisotropies (17). For two chemically identical donor-acceptor pairs, characterized by the same
Ro (the same
2,
d, and
J), the differences in the transfer efficiencies, E1 and E2, result
exclusively from the different distances between the donor and
acceptor, R1 and R2. The
relative ratio of the two distances is then defined by using Equation 10 as follows.
|
(Eq. 11)
|
Determination of Rigorous Thermodynamic Binding Isotherms and
Absolute Stoichiometries of the DnaB Helicase-ssDNA Complexes--
In
this work, we followed the binding of the DnaB protein to the ssDNA
oligomers by monitoring the fluorescence increase,
F, of
ssDNA etheno-derivatives upon the complex formation. Proteins and
nucleic acids may form complexes characterized by different spectroscopic properties, particularly when multiple ligand binding processes are studied. In applying spectroscopic methods to monitor the
ligand macromolecule interactions, one should not assume strict proportionality between the observed signal change and the degree of
binding unless the existence of such proportionality has been shown
(15). The general method to obtain thermodynamically rigorous estimates
of the average degree of binding of the protein per ssDNA oligomer,

i, and the free protein concentration, PF, has been previously described by us (8, 15, 25).
Briefly, the experimentally observed
F has a contribution from each of the different possible "i" complexes of the
DnaB hexamer with a nucleic acid. Thus, the observed fluorescence
increase is functionally related to 
i by the
equation,
|
(Eq. 12)
|
where
Fimax is
the molecular parameter characterizing the maximum fluorescence
increase of the nucleic acid with the DnaB protein bound in complex
i. The same value of
F, obtained at two
different total nucleic acid concentrations,
NT1 and
NT2, indicates the same
physical state of the nucleic acid, i.e. the degree of
binding, 
i, and the free DnaB protein concentration,
PF, must be the same. The value of 
i
and PF is then related to the total protein concentrations, PT1 and
PT2, and the total
nucleic acid concentrations,
NT1 and
NT2, at the same value of
F, by the following equations,
|
(Eq. 13)
|
|
(Eq. 14)
|
where x = 1 or 2 (12, 20).
 |
RESULTS |
Micrococcal Nuclease Digestion Reveals Large Structural
Heterogeneity within the DNA Binding Site of the E. coli DnaB
Helicase--
Quantitative fluorescence titrations and
photo-cross-linking experiments, using ssDNA oligomers, showed that the
DnaB hexamer has a single ssDNA binding site encompassing 20 ± 3 nucleotide residues and located predominantly on a single subunit
(12-14). The first evidence of the structural heterogeneity within the DNA binding site came from nuclease digestion-protection studies of the
DNA in the complex with the helicase. In the first set of experiments,
the complex of the DnaB hexamer with the 20-mer dT(pT)19
labeled at its 5'-end with 32P in the presence of 1 mM AMP-PNP was subjected to micrococcal nuclease digestion
as a function of time. The protein was in molar excess over the 20-mer
to ensure complete saturation of the nucleic acid. Fig.
1a shows the polyacrylamide
sequencing gel of dT(pT)19 after digestion with the
nuclease, at different time intervals, in the absence and presence of
the helicase. In the absence of the helicase, in our solution
conditions, the 20-mer was digested within 20 min. A dramatically
different behavior was observed in the presence of the enzyme. The
digestion process was less efficient, indicating significant protection
of the nucleic acid against the nuclease by the enzyme. Moreover, at
prolonged digestion times, a nucleic acid fragment of 10 or 11 nucleotide residues was strongly protected by the helicase. At the
longest times, this was the major nucleic acid fragment on the gel,
resistant to further nuclease action (Fig. 1a).

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Fig. 1.
a, autoradiogram of the 15% sequencing
polyacrylamide gel electrophoresis of the
5'-[32P](dT)20 and DnaB
protein-5'-[32P](dT)20 complexes, after
micrococcal nuclease (MN) digestion in buffer T2 (pH 8.1, 4 °C) containing 100 mM NaCl, 1 mM
CaCl2, and 1 mM AMP-PNP. The concentration of
the protein and the 20-mer are 1 × 10 6
M (hexamer) and 5 × 10 7 M
(oligomer). Oligomers of different lengths are included in lane
1 as molecular markers. Lanes 2-6 show the
different digestion times without the DnaB helicase. Lane 2,
5'-[32P](dT)20, 0 s; lane 3,
30 s; lane 4, 60 s; lane 5, 300 s;
lane 6, 900 s; lane 7, 1800 s.
Lanes 8-14 show the complex
5'-[32P](dT)20-DnaB helicase at different
digestion times. Lane 8, 30 s; lane 9,
60 s; lane 10, 180 s; lane 11, 300 s; lane 12, 600 s; lane 13, 900 s;
lane 14, 1800 s. b, autoradiogram of the
15% sequencing polyacrylamide gel electrophoresis of the
5'-[32P](dA)70 and DnaB
protein-5'-[32P](dA)70 complexes, after
micrococcal nuclease digestion in buffer T2 (pH 8.1, 4 °C)
containing 100 mM NaCl, 1 mM CaCl2
and 1 mM AMP-PNP. The concentration of the protein and the
70-mer are 1 × 10 6 M (hexamer) and
5 × 10 7 M (oligomer), respectively.
Lanes 2-7 show the 5'-[32P](dA)70
at different digestion times without the DnaB helicase. Lane
2, 5'-[32P](dA)70, 0 s; lane
3, 30 s; lane 4, 60 s; lane 5,
300 s; lane 6, 900 s; lane 7, 1800 s. Lanes 8-14 show the complex
5'-[32P](dA)70-DnaB helicase at different
digestion times. Lane 8, 30 s; lane 9,
60 s; lane 10, 180 s; lane 11, 300 s; lane 12, 600 s; lane 13, 900 s;
lane 14, 1800 s.
|
|
The size of the protected fragment was not dependent upon the length or
type of base of the oligomer bound to the DnaB protein, indicating that
protection against the nuclease digestion is limited to the nucleic
acid bound within the single DNA binding site of the helicase. Fig.
1b shows polyacrylamide sequencing gels of dA(pA)69 after digestion with the nuclease, at different
time intervals, and in the absence and presence of the helicase. As in
the case of dT(pT)19, the only predominant oligomer
protected by the helicase in the complex with dA(pA)69,
after prolonged digestion, is a ssDNA fragment, 10 or 11 nucleotide
residues long.
These data indicate that, within the total DNA binding site of the DnaB
helicase, approximately half of the ~20 nucleotide residues occluded
by the helicase are bound differently than the remaining half,
resulting in the observed nuclease digestion pattern. Thus, these
results indicate that the total DNA binding site of the DnaB helicase
is built of two structurally and possibly functionally different
binding subsites (see below).
Binding of 10-mer, d
A(p
A)9, to the DnaB
Helicase--
To determine whether or not there is a difference in
affinities between the two subsites of the total DNA binding site of the helicase that could result in their different functional roles in
the enzyme activities, we performed quantitative thermodynamic studies
of the binding of a ssDNA oligomer containing only 10 residues to the
DnaB hexamer. These are the partial nucleic acid ligands that can only
interact with half of the total binding site of the enzyme.
Fluorescence titrations of d
A(p
A)9 with the DnaB
helicase, at five different nucleic acid concentrations, in buffer T2
(pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM AMP-PNP, are shown in Fig.
2a. As the nucleic acid
concentration increases, the same relative fluorescence increase is
reached at higher DnaB protein concentrations. The selected nucleic
acid concentrations provide the separation of binding isotherms up to a
relative fluorescence increase of ~4.1, with the plateau at the
maximum relative fluorescence increase,
Fmax = 4.3 ± 0.2. To obtain thermodynamically rigorous binding
parameters, independent of any assumption about the relationship between the observed signal and the degree of binding, 
i, the fluorescence titration curves shown in Fig. 2a were
analyzed, using the approach outlined under "Materials and
Methods." Fig. 2b shows the dependence of the observed
relative fluorescence increase as a function of the average number of
the DnaB hexamers bound per oligomer. The plot is linear, indicating
that, in the studied binding density range, there is a very similar
enhancement of the nucleic acid fluorescence upon the binding of the
oligomer to the DnaB protein. The value of 
i could be
determined up to ~90% of the observed signal change. Short
extrapolation to the maximum value of the fluorescence increase
provides the stoichiometry of the complex. Thus, the data show that
only one 10-mer strongly binds to the DnaB hexamer, indicating that the structural differences between the subsites, resulting in protection of
~10 nucleotide residues of ssDNA in the total DNA binding site against nuclease digestion, are reflected in the large differences in
the affinities between the two DNA binding subsites. The solid lines in Fig. 2a are computer fits of the binding
isotherms to a single binding site that provide the binding constant
K = (1.7 ± 0.3) × 107
M
1. Comparison with the binding constant for
the 20-mer, d
A(p
A)19, previously obtained by us in
the same solution conditions (K = 3 × 107 M
1), shows that the 10-mer
binds with an affinity very similar to the 20-mer (13). Thus, the data
indicate that the predominant part of the free energy of binding the
DnaB helicase to ssDNA comes from the interactions of the nucleic acid
with the strong binding subsite of the enzyme (see
"Discussion").

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Fig. 2.
a, fluorescence titrations of
d A(p A)9 with the DnaB protein monitored by the
increase of the nucleic acid fluorescence in buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP, at three different nucleic acid concentrations (oligomer):
4.5 × 10 7 M ( ); 9.0 × 10 7 M ( ); 1.9 × 10 6
M ( ); 3.6 × 10 6 M ( );
8.0 × 10 6 M ( ). Solid
lines are computer fits of the single-site binding isotherm,
F = Fmax
(KPF/(1 + KPF)), with intrinsic
binding constant K1 = 1.7 × 107 M 1 and
Fmax = 4.3. b, the dependence of
the relative increase of the d A(p A)9 fluorescence
upon the average number of DnaB helicase hexamers bound per oligomer
( ). The absolute value of the average number of DnaB helicase
hexamers bound per oligomer,  i, has been determined using
the thermodynamically rigorous approach described under "Materials
and Methods." The solid line is a computer fit using the
single-site binding isotherm ( F = Fmax
(K1PF/(1 + K1PF));  i = K1PF/(1 + K1PF)) with
K1 = 1.7 × 107
M 1 and Fmax = 4.3;
PF is the free d A(p A)9
concentration.
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|
Quantitative fluorescence titrations at a very high concentration of
d
A(p
A)9 (~8 × 10
6 M
(oligomer)) did not show detectable binding of the additional 10-mer
(Fig. 2a). Titrations at higher nucleic acid concentrations are very difficult because they require very high concentrations of
stock solutions of the DnaB protein, which are beyond the attainable solubility of the protein. Therefore, we used the analytical
centrifugation technique to assess the affinity of DNA to the second
subsite. In these experiments, we used a 10-mer, dA(pA)9,
labeled at the 5'- or 3'-end with fluorescein (see "Materials and
Methods"). This approach allowed us to monitor exclusively the
nucleic acid and the protein-nucleic acid complex without the
interference of the protein and AMP-PNP absorbance. The sedimentation
velocity profiles (monitored at 515 nm) of the DnaB
helicase-dA(pA)9-3'-Fl mixture at the nucleic acid and
helicase concentrations of 9 × 10
5 and 2 × 10
5 M, respectively, in buffer T2 (pH 8.1, 20 °C) containing 100 mM NaCl and 1 mM
AMP-PNP, are shown in Fig. 3. The
sedimentation run was performed at 60,000 rpm. It is clear that,
initially, two independently moving boundaries exist. The slow moving
boundary has a sedimentation coefficient of
s20,w = 1.4 ± 0.2, which is the
s20,w value of the free
dA(pA)9-3'-Fl. The fast moving boundary contains
dA(pA)9-3'-Fl in the complex with the DnaB helicase. We
have previously shown that the DnaB hexamer fully preserves its
hexameric structure in the complex with the ssDNA (8, 13). After the
fast moving boundary reaches the cell bottom, only the slow moving
boundary of the free dA(pA)9-3'-Fl still remains
(dashed lines). Notice that during the sedimentation process, the boundary of the complex migrates in the field of the
constant free 10-mer concentration [T]Free
1/K1, thus assuring that the enzyme always has
the strong binding subsite saturated with the nucleic acid. At 515 nm,
one monitors exclusively the total concentration of the 10-mer.
Comparison of the contributions of the slow and fast moving boundaries
with the total absorption of the sample shows that 36% of the total
nucleic acid concentration migrated in the fast moving boundary (Fig.
3). From the known total concentration of the DnaB helicase in the
sample, the stoichiometry of the complex is calculated to be 1.6 ± 0.2, which indicates that at this 10-mer concentration we observed
significant saturation (60%) of the second DNA binding subsite.

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Fig. 3.
Absorption profiles at 515 nm of the
sedimentation velocity runs of the dA(pA)9-3'-Fl-DnaB
protein complex in buffer T2 (pH 8.1, 20 °C) containing 100 mM NaCl and 1 mM AMP-PNP, at a 4.5:1 molar
excess of the nucleic acid over the enzyme. The concentration of
the DnaB hexamer is 2 × 10 5 M
(hexamer), and the concentration of the dA(pA)9-3'-Fl is
9 × 10 5 M (oligomer). Solid
lines are initial scans of the samples, which include slow and
fast moving boundaries of the nucleic acid and the complex,
respectively. Dashed lines are scans of the sample after the
fast moving boundary reached the bottom of the cell. The initial part
of the last scan indicates the location of the base line (time interval
was 8 min; 60,000 rpm).
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Because we know the free nucleic acid concentration from the absorbance
of the slowly moving boundary, we can estimate the macroscopic ssDNA
binding constant for the second subsite. In the considered case, the
first binding subsite of the helicase is completely saturated with the
10-mer. Therefore, the partition function of the system, Z,
and the degree of binding to the second subsite,
2, are
as follows,
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(Eq. 15)
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(Eq. 16)
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and K2 is defined as follows.
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(Eq. 17)
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Introducing the values of
2 = 0.6 and
[T]Free = 5.8 × 10
5 M,
obtained from the sedimentation velocity experiments, provides the
value of K2 = (2.6 ± 1) × 104
M
1. A similar value of the binding constant
K2 of the dA(pA)9-3'-Fl and
5'-Fl-dA(pA)9 for the weak binding subsite has been
obtained using lower and higher concentrations of the nucleic acids.
Thus, the data show that the affinity of ssDNA for the second subsite is ~3 orders of magnitude lower than the affinity for the strong binding subsite.
Interactions of ssDNA Oligomers Having Different Lengths with the
Strong ssDNA Binding Subsite--
To obtain further insight into the
interactions of the DNA in the strong binding subsite, we performed
quantitative fluorescence titrations of a series of ssDNA oligomers of
different lengths. Fluorescence titrations of
d
A(p
A)8, d
A(p
A)7,
d
A(p
A)6, d
A(p
A)5, d
A(p
A)4, and d
A(p
A)3, with the DnaB
helicase in buffer T2 (pH 8.1, 10 °C) containing 100 mM
NaCl and 1 mM AMP-PNP, are shown in Fig.
4a. For comparison, the
fluorescence titration of the 10-mer, d
A(p
A)9, with
DnaB is also included. With the decreasing number of residues, the
relative maximum fluorescence changes, and the affinity decreases. In
the case of 9- and 8-mers, the maximum fluorescence change,
Fmax, upon saturation with the helicase, is
still similar to the one determined for the 10-mer. However, the
affinity is lower than the affinity of the 10-mer and is characterized by binding constants K9 = (1.5 ± 0.5) × 107 M
1 and
K8 = (8 ± 2) × 106
M
1, respectively. A dramatic drop in the
affinity and maximum relative fluorescence increase is observed in the
case of the 7- and 6-mer (Fig. 4a; see Table
I). No detectable binding to the helicase occurs in the case of 5- and 4-mers (Fig. 4a). Titrations at
very high concentrations of the 5- and 4-mer could only provide a
semiquantitative estimate of the affinities, due to the required DnaB
concentration beyond the solubility of the protein; however, these
experiments indicate that the binding constants for the 5- and 4-mers
are not higher than 1 × 104
M
1 (data not shown). Fig. 4b shows
the dependence of the natural logarithm of binding constants of studied
oligomers to the DnaB protein as a function of the number of nucleotide
residues in the ssDNA oligomer. The plot is nonlinear, a clear
indication that the affinity is not a simple function of the length of
the nucleic acid. The difference of the 2 residues between 10-mer and
8-mer causes only an ~0.3 kcal/mol decrease of the free energy of
interactions. The difference in the 2 residues between 7-mer and 5-mer
decreases the free energy of binding by at least ~3 kcal/mol,
practically eliminating the binding of the 5-mer to the enzyme in
studied solution conditions. These data show that, to efficiently bind
to the strong DNA binding subsite, the nucleic acid must span 6 or 7 residues. Thus, the results indicate a complex structure of the ssDNA
strong binding subsite where the direct contacts between the helicase
and the nucleic acid, decisive in complex formation, are separated by 6 or 7 nucleotides (see "Discussion").

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Fig. 4.
a, fluorescence titrations of
d A(p A)8, d A(p A)7,
d A(p A)6, d A(p A)5,
d A(p A)4, and d A(p A)3, with the DnaB
protein monitored by the increase of the nucleic acid fluorescence in
buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM AMP-PNP. Concentrations of all oligomers are 4.5 × 10 7 M (oligomer). ,
d A(p A)8; , d A(p A)7; ,
d A(p A)6; , d A(p A)5; +,
d A(p A)4; , d A(p A)3. For
comparison, the fluorescence titration of d A(p A)9 is
also included ( ). Solid lines are computer fits of the
single-site binding isotherm, F = Fmax(K1PF/(1 + K1PF)), with binding
constants K1 and Fmax
as follows: 1.5 × 107 M 1
and 4.3; 8 × 106 M 1 and
4.3; 3.3 × 106 M 1 and 3.3;
and 4 × 105 M 1 and 2.6 for
the 9-, 8-, 7-, and 6-mer, respectively. b, the dependence
of the natural logarithm of binding constant as a function of the
length of the oligomer bound to the strong subsite of the DnaB helicase
( ). The binding constants for 5- and 4-mers have an assigned maximum
value at 1 × 104 M 1, which
is below the minimum affinity detectable in our fluorescence titrations
(~5 × 104 M 1), although
the affinities of these oligomers could be characterized by even lower
binding constants, as indicated by the error bars.
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Table I
Thermodynamic and fluorescence parameters of ssDNA oligomer binding to
the DnaB helicase in buffer T2 (pH 8.1, 100 mM NaCl, 1 mM AMP-PNP, 10 °C; ex = 325 nm, em = 410 nm)
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Salt Effect on the Affinity of the DnaB Helicase to ssDNA
Oligomers--
Fluorescence titrations of d
A(p
A)8
with the DnaB helicase in buffer T2 (pH 8.1, 10 °C), containing 1 mM AMP-PNP and different NaCl concentrations, are shown in
Fig. 5a. As the salt
concentration increases, the isotherms shift toward higher total DnaB
protein concentrations, indicating a decreasing affinity of the
protein-nucleic acid complex at higher salt concentrations. It should
also be noted that
Fmax is lower at higher
salt concentrations, decreasing from 4.3 ± 0.2 at 100 mM to 2.8 ± 0.2 at 407 mM [NaCl]. A
similar decrease of the maximum fluorescence increase upon the helicase binding has been observed for all other oligomers (data not shown).

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Fig. 5.
a, fluorescence titrations of
d A(p A)9 with the DnaB protein in buffer T2 (pH 8.1, 10 °C) containing 1 mM AMP-PNP, at different NaCl
concentrations as follows. , 100 mM; , 194 mM; , 304 mM; , 407 mM.
Solid lines are computer fits using single-site binding
isotherm, F = Fmax(K1PF/(1 + K1PF)), with
Fmax and K1 as
follows. , 4.3 and 1.5 × 107
M 1; , 3.7 and 7 × 106
M 1; , 3.3 and 3 × 106
M 1; , 2.8 and 1.3 × 106
M 1. b, the dependence of the
intrinsic binding constant K1 for the binding of
ssDNA oligomers of different lengths to the strong binding subsite of
DnaB helicase upon NaCl concentrations in solution (log-log plots) in
buffer T2 (pH 8.1, 10 °C) containing 1 mM AMP-PNP. ,
d A(p A)9; , d A(p A)8; ,
d A(p A)7; , d A(p A)6; ,
d A(p A)5.
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The dependence of the logarithm of the intrinsic binding constants for
10-, 9-, 8-, 7-, and 6-mers upon the logarithm of [NaCl] (log-log
plot) is shown in Fig. 5b. Within experimental accuracy, the
plots are linear in the studied salt concentration ranges, which is
different from the nonlinear behavior of the log-log plot previously
determined in the case of the 20-mer, d
A(p
A)19 (12,
13). With increasing salt concentrations, the affinities of all
oligomers decrease, indicating that the binding process is accompanied
by a net release of ions with the slopes
logK/
log[NaCl] =
1.4 ± 0.4,
1.5 ± 0.3,
1.4 ± 0.4,
1.5 ± 0.4, and
1.4 ± 0.4 for
10-, 9-, 8-, 7-, and 6-mer, respectively (27) (Table I). Thus, these
data indicate that a similar number of ~1.5 ions is released upon the
complex formation with each of the oligomers being long enough to
provide all essential contacts with the enzyme in the binding
subsite.
Previously, we determined that binding of a 20-mer,
d
A(p
A)19, which spans the entire total DNA binding
site, to the DnaB helicase is accompanied by the maximum release of
~3.7 ions (13). This number is significantly higher than the ~1.4
obtained for the 10-mer (Table I). This comparison suggests that the
interactions of ssDNA with the weak binding subsite are accompanied by
a net release of ~two ions. Another possibility is that interactions between the strong and weak subsites, simultaneously saturated with
nucleic acid in the complex with d
A(p
A)19, result in
the net release of ~two additional ions. At present, we cannot
exclude either of these possibilities.
Determination of the Orientation of the E. coli DnaB Helicase with
Respect to the Polarity of the Sugar-Phosphate Backbone of ssDNA, Using
the Fluorescence Energy Transfer Method--
Determination of the
mutual orientations of proteins and nucleic acids in the complex should
be based on a method that is sensitive to the differences in distances
between different, specific regions of both macromolecules (17, 22).
Fluorescence energy transfer between a donor and an acceptor, placed in
specific locations on a protein and a nucleic acid, provides a very
sensitive technique to assess the relative proximities between
different regions of both macromolecules in the complex. The
orientation of the DnaB helicase, in the complex with ssDNA, was
determined by using the 20-mer, dT(pT)19, labeled with
fluorescein (acceptor) at its 5'- or 3'-end, respectively, and the DnaB
protein variant, R14C, specifically labeled with a coumarin derivative
(donor), CPM, at the small 12-kDa domain of the enzyme (see
"Materials and Methods"). If the DnaB helicase binds predominantly
in a single orientation, with respect to the polarity of the
sugar-phosphate backbone of ssDNA, then different responses of the
donor and acceptor fluorescence should be observed, depending on the
different location of the acceptor on the nucleic acid.
The elongated DnaB protein monomer is built of two structural domains,
small 12-kDa and large 33-kDa domains connected at the "hinge"
region (28) as visualized by electron microscopy data (10, 11). In the
hexamer, all protomers are oriented with their small 12-kDa domains in
the same direction (10, 11). Because the protein does not have natural
cysteines, we replaced arginine at position 14 from the N terminus of
the protein located in the small 12-kDa domain of the enzyme with a
single cysteine residue, using site-directed mutagenesis. Subsequently,
this cysteine residue was specifically modified with CPM to provide
R14C-CPM (see "Materials and Methods"). The selection of the
modification site was directed by the fact that removal of the entire
14-amino acid fragment from the N terminus of the protein did not
affect, to any extent, the biological functions of the protein (28). As
a result of modification, the R14C DnaB hexamer has six CPM molecules
located in the small domain of each protomer (R14C-CPM). Thus, 6 CPM
residues form a ring at one end of the DnaB hexamer.
The emission spectrum of R14C-CPM strongly overlaps the absorption
spectrum of the fluorescein. These spectroscopic properties of CPM make
the marker an excellent fluorescence donor for fluorescein (29). The
presence of unlabeled dT(pT)19 causes very little change in
the fluorescence emission spectra of R14C-CPM (
ex = 435 nm); however, the presence of R14C-CPM causes an ~2-fold decrease of
the fluorescence intensity of 5'-Fl-dT(pT)19
(
ex = 485 nm), although with the excitation at 485 nm
only fluorescein on the 5'-Fl-dT(pT)19 absorbs light (data
not shown). Saturation of the 20-mer with the unlabeled DnaB protein
causes only an ~8% decrease of the 5'-Fl-dT(pT)19
fluorescence (data not shown). It is evident that, even in the absence
of the energy transfer process, the presence of 6 hydrophobic CPM
residues affects the quantum yield of fluorescein at the 5'-end of the
ssDNA, which already suggests close proximity between the CPMs and
fluorescein. The quantum yield of fluorescein is independent of the
excitation wavelength between 400 and 500 nm (22). Thus, as expected,
the ratio of quantum yields of 5'-Fl-dT(pT)19 in the
complex with R14C-CPM and free in solution,
BA/
FA,
is constant and equal to 0.51 over a tested range of excitation wavelengths between 465 and 500 nm. In this spectral range of excitation, no detectable fluorescence energy transfer from CPM residues to fluorescein occurs. Thus, this ratio of quantum yields, independent of excitation wavelength, reflects the change of the emission intensity of 5'-Fl-dT(pT)19, resulting exclusively
from the formation of the complex with R14C-CPM, in the absence of the
energy transfer process, and can be used to obtain the spectrum of
5'-Fl-dT(pT)19, in the presence of R14C-CPM, without the
changes induced by the energy transfer process at any excitation
wavelength (Equations 7 and 8).
The dashed line in Fig.
6a is the sum of the emission
spectra (
ex = 435 nm) of the R14C-CPM and
5'-Fl-dT(pT)19 in the presence of unlabeled nucleic acid
and R14C-CPM (without energy transfer), respectively, in buffer T2 (pH
8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP. The solid line is the fluorescence emission spectrum of the complex of R14C-CPM with 5'-Fl-dT(pT)19 at
the same concentrations of the protein and nucleic acid as in the case
of the sum of independent components of the complex. Clearly, there is
a dramatic difference between the sum of the independent donor and
acceptor spectra and the spectrum where both donor and acceptor are
placed in the same complex. The emission intensity of R14C-CPM at 476 nm in the complex with 5'-Fl-dT(pT)19 is decreased by ~ 35%, as compared with the R14C-CPM complexed with unlabeled dT(pT)19. The decrease of emission at 476 nm, where there
is no contribution from fluorescein emission, indicates significant fluorescence energy transfer from the CPM residues located on the small
12-kDa domains of the DnaB hexamer to the fluorescein moiety placed at
the 5'-end of the bound 5'-Fl-dT(pT)19.

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Fig. 6.
a, sum of the fluorescence emission
spectra (- - -) of DnaB R14C-CPM in the presence of unlabeled
dT(pT)19 (4.5 × 10 7 M
(oligomer)) and 5'-Fl-dT(pT)19 in the presence of R14C-CPM
(without energy transfer) ( ex = 435 nm) in buffer T2 (pH
8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP and the fluorescence emission spectrum of the complex of
R14C-CPM with 5'-Fl-dT(pT)19 ( ex = 435 nm)
( ) in the same buffer. Concentrations of 5'-Fl-dT(pT)19
and the protein were 4.5 × 10 7 M
(oligomer) and 9.6 × 10 7 M (hexamer),
respectively. The fluorescence emission spectrum of R14C-CPM normalized
at 476 nm (peak) to the emission spectrum of the protein in the complex
with 5'-Fl-dT(pT)19 (- · - · -) is also
included. b, sensitized emission spectrum of
5'-Fl-dT(pT)19 ( ex = 435 nm) in the complex
with R14C-CPM ( ), obtained after subtraction of the normalized
spectrum of R14C-CPM (see Fig. 7a) in buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP superimposed on the fluorescence emission spectrum of
5'-Fl-dT(pT)19 in the presence of R14C-CPM (without energy
transfer) (- - -) obtained at the same excitation wavelength by
multiplying the spectrum of free, labeled 20-mer by the quantum yield
ratio,
BA/ FA = 0.51. Concentrations of 5'-Fl-dT(pT)19 and R14C-CPM are
4.5 × 10 7 M (oligomer) and 9.6 × 10 7 M (hexamer), respectively.
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Comparison between the sum of the spectra of independent components of
the complex and the spectrum of the complex in Fig. 6a shows
that the fluorescence intensity of the fluorescein residue of
5'-Fl-dT(pT)19, with the peak at 520 nm, is strongly
increased in the complex with R14C-CPM (
ex = 435 nm).
Recalling that fluorescein does not contribute to the CPM emission band
at 476 nm, we can normalize the spectra of R14C-CPM-unlabeled
dT(pT)19 and R14C-CPM-5'-Fl-dT(pT)19 complex at
476 nm. The difference between the normalized spectrum of
R14C-CPM-unlabeled dT(pT)19 and the spectrum of the complex R14C-CPM-5'-Fl-dT(pT)19 provides the sensitized emission
spectrum of the 5'-Fl-dT(pT)19 bound to R14C-CPM. The
emission spectrum of 5'-Fl-dT(pT)19 in the complex with
R14C-CPM, without energy transfer, with the sensitized emission
spectrum of 5'-Fl-dT(pT)19 is shown in Fig. 6b.
It is evident that in the presence of the donor, CPM, the fluorescence
intensity of the fluorescein at the 5'-end of the 20-mer is increased
by ~220%.
Analogous experiments were performed with a 20-mer,
dT(pT)19-Fl-3', having fluorescein located at the opposite
3'-end of the nucleic acid. Unlike the case of
5'-Fl-dT(pT)19, formation of the complex with R14C-CPM
causes only an ~8% decrease of the fluorescence of
dT(pT)19-Fl-3' (
ex = 485 nm), which is the
same as observed in the presence of unlabeled protein (data not shown).
This difference results from the larger distance between CPM residues
on the small 12-kDa domains of the DnaB hexamer and fluorescein at the
3'-end of the 20-mer (see below). The dashed line in Fig.
7a is the sum of the
fluorescence emission spectra of independent components of the complex,
R14C-CPM in the presence of unlabeled dT(pT)19, and the
fluorescence emission spectrum of dT(pT)19-Fl-3' in the presence of R14C-CPM (without energy transfer), in buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP (
ex = 435 nm). The solid line in Fig.
7a is the fluorescence emission spectrum the R14C-CPM and
dT(pT)19-Fl-3' complex at the same concentrations of the
protein and nucleic acid as independent components of the complex.
Contrary to the situation with 5'-Fl-dT(pT)19, only a small
difference is observed when both the donor, CPM on the DnaB protein,
and the acceptor, fluorescein on the 3'-end of the 20-mer, are placed
in the same complex as compared with the sum of the spectra of
independent components of the complex. The emission intensity of
R14C-CPM is only decreased by ~11% as compared with ~35% observed
for R14C-CPM with 5'-Fl-dT(pT)19, indicating a very diminished fluorescence energy transfer from CPM to the fluorescein moiety, when the acceptor is located at the 3'-end of the
dT(pT)19. Also, the sensitized emission of the fluorescein
located at the 3'-end of the 20-mer is only increased by ~43% as
compared with ~220% in the complex of R14C-CPM with
5'-Fl-dT(pT)19 (Fig. 6b).

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Fig. 7.
a, sum of the fluorescence emission
spectra (- - -) of R14C-CPM in the presence of unlabeled
dT(pT)19 (4.5 × 10 7 M
(oligomer)) and dT(pT)19-Fl-3' in the presence of R14C-CPM
(without energy transfer) ( ex = 435 nm) in buffer T2 (pH
8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP and the fluorescence emission spectrum of the complex of
R14C-CPM with dT(pT)19-Fl-3' ( ex = 435 nm)
in the same solution conditions ( ). Concentrations of
dT(pT)19-Fl-3' and R14C-CPM are 4.5 × 10 7 M (oligomer) and 9.6 × 10 7 M (hexamer), respectively. The
fluorescence emission spectrum of R14C-CPM normalized at 476 nm (peak)
to the emission spectrum of R14C-CPM in the complex with
dT(pT)19-Fl-3' (- · - · -) is also included.
b, sensitized emission spectrum of
dT(pT)19-Fl-3' ( ex = 435 nm) in the complex
with R14C-CPM ( ) obtained after subtraction of the normalized
spectrum of R14C-CPM in buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM AMP-PNP superimposed on the
fluorescence emission spectrum of dT(pT)19-Fl-3' in the
presence of R14C-CPM (without energy transfer) (- - -), obtained at
the same excitation wavelength. Concentrations of
dT(pT)19-Fl-3' and R14C-CPM are 4.5 × 10 7 M (oligomer) and 9.6 × 10 7 M (hexamer), respectively.
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The dramatic difference between the emission spectrum of the complex of
R14C-CPM with 5'-Fl-dT(pT)19 and the spectrum of the complex with dT(pT)19-Fl-3' clearly shows that the helicase
binds ssDNA in a predominantly single orientation, with respect to the polarity of the ssDNA sugar-phosphate backbone. If the helicase could
bind ssDNA in two different orientations with equal probability, then
the changes in the spectra of the complexes with the 20-mer, labeled
with fluorescein at the 5'- or 3'-ends, would be indistinguishable.
The effect of the location of the fluorescence acceptor on the observed
spectral properties of the studied complexes is reflected in the large
differences in the true energy transfer efficiencies, E.
Using Equations 5 and 8, we obtained the apparent transfer efficiencies
of ED = 0.77 ± 0.05 and EA = 0.55 ± 0.03, respectively, for the complex of R14C-CPM with
5'-Fl-dT(pT)19. This difference between
ED and EA indicates that
fluorescein, at the 5'-end of the bound dT(pT)19, induces some additional nondipolar CPM fluorescence quenching. The true Förster fluorescence transfer efficiency from CPM, located on the
small 12-kDa domain to the fluorescein residue at the 5'-end of
dT(pT)19, is then described by Equation 9, which yields
E = 0.71 ± 0.05. Analogous calculations of the
fluorescence energy transfer efficiency in the complex of R14C-CPM with
dT(pT)19-Fl-3' yield ED = 0.18 and
EA = 0.09 ± 0.01 (Table II). In this case, the true Förster
transfer efficiency is E = 0.1 ± 0.01. The large
difference between the true energy transfer efficiencies shows that the
5'-end of the 20-mer, dT(pT)19, is in much closer proximity
to the CPM residues, which are located on the small domains of the DnaB
hexamer, than to the 3'-end of the nucleic acid (see
"Discussion").
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Table II
Fluorescence properties of 5'-Fl-dT(pT)19 and
dT(pT)19-Fl-3' in the complex with the E. coli DnaB helicase
and the DnaB helicase variant modified with CPM (R14C-CPM) in buffer T2
(pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM AMP-PNP
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The determination of exact distances between the donors (CPM) and
acceptors (fluorescein) is beyond the scope of the present discussion
on the mutual orientation between the DnaB helicase and the ssDNA in
the complex. However, using Equation 11 we can estimate the approximate
ratio of the distances between the 5'- and the 3'-end of the
dT(pT)19 oligomer from the center of the mass of CPM donors
located on the small domains of the DnaB hexamer. Introducing
E1 = 0.71 and E2 = 0.1 into Equation 11, we obtained R1/R2 = 0.60. Thus, the
average distance of the 5'-end of the 20-mer is only 60% of the
distance between the donors and the 3'-end of the nucleic acid.
Very similar behavior to the one described above has been observed when
different donor-acceptor pairs have been
used.3 These results show,
for the first time, that the DnaB hexamer binds ssDNA in a single
orientation, with respect to the sugar-phosphate backbone of the
nucleic acid. In the complex, the small 12-kDa and the large 33-kDa
domains of the enzyme face the 5'- and 3'-ends of the nucleic acid,
respectively.
DNA Mobility within the Strong and Weak DNA Binding Subsite of the
DnaB Helicase--
Assessment of the relative mobility of the
different segments of the nucleic acid, within the DNA binding site,
can be obtained by measuring the emission anisotropy of the fluorescent
markers placed in different locations on the nucleic acid. To determine the relative mobility of ssDNA in two subsites of the total DNA binding
site of the DnaB helicase, we determined the emission anisotropy of
5'-Fl-dT(pT)19 and dT(pT)19-Fl-3' in the
complex with the helicase. Anisotropies of both samples are constant
across their emission spectra, indicating the lack of a significant
local heterogeneity around the fluorescent markers (spectra not shown). However, the anisotropy of 5'-Fl-dT(pT)19,
r = 0.28 ± 0.01, is significantly higher than the
anisotropy, r = 0.24 ± 0.01, determined for
dT(pT)19-Fl-3'. Because the fluorescence lifetimes of
fluorescein in both complexes are very similar (~4 ns, data not
shown), the obtained data indicate significantly higher mobility of the
nucleic acid at its 3'-end.
Analogous fluorescence energy transfer and anisotropy studies with a
10-mer, dA(pA)9, labeled with fluorescein at the 5'- or
3'-ends of the 10-mer indicate that its 5'-end is located in close
proximity to the 12-kDa domain of the enzyme and has a similar strong
decrease in its mobility (data not shown). As we described above, this
oligomer binds exclusively to the strong subsite in the DNA binding
site of the DnaB helicase. Thus, fluorescence energy transfer and
anisotropy data indicate that the nucleic acid binds with the first 10 nucleotides from its 5'-end to the strong DNA binding subsite of the
total DNA binding site of the helicase.
 |
DISCUSSION |
The Total DNA Binding Site of a Helicase--
Helicases play a key
role in all aspects of DNA metabolism, and this role is related to the
interactions of the enzyme with ssDNA and dsDNA controlled by binding
and hydrolysis of a nucleoside triphosphate, e.g. ATP (30).
Understanding the functional and structural aspects of the DNA binding
site is a prerequisite for our understanding of how the enzymes perform
their functions. Yet, little is known about the structure of the DNA
binding site of any hexameric helicase and the functional
interrelations within the binding site. In this work, we provide the
first insight into the complex structure/function relationship of the
DNA binding site of a hexameric replicative helicase, the E. coli DnaB protein.
Our previous studies with polymer ssDNA and ssDNA oligomers showed that
in a stationary complex with the ATP-nonhydrolyzable analog, AMP-PNP,
the enzyme has a single binding site located on a single subunit of the
hexamer (12-14). Additionally, this single binding site is used when
the enzyme binds to the DNA substrates resembling the replication fork
(8, 13, 25, 26). These results indicate that the observed single
binding site is, in fact, the total DNA binding site of the enzyme
that, in functional complexes on the junction between ssDNA and dsDNA
with the replication fork, encompasses both single- and double-stranded
conformations of nucleic acid over a stretch of ~20 nucleotide
residues.
The operational definition of the total binding site of the enzymes,
which perform their catalysis on polymer lattices, such as helicases,
should refer to the complex of the enzymes with a polymer substrate. A
total binding site of an enzyme is used as a single entity that
interacts with a continuous stretch of polymer substrate. This
continuous fragment of the polymer substrate (DNA), within the total
binding site, defines the site size of the enzyme-nucleic acid complex.
The total binding site can be heterogeneous, i.e. built of
functionally and/or structurally different areas, subsites, specific
for the catalytic functions of the enzyme. However, the location of the
subsites is sequential, i.e. they are placed along the
polymer substrate. The total binding site can perform the dominant
catalytic process characteristic for the enzyme, e.g.,
unwinding of the duplex DNA. Such a binding site can be located on a
single subunit of an oligomeric enzyme, such as the DnaB helicase;
thus, there may be several total binding sites, but only one site (one
subunit) at a time is engaged in interactions with DNA during the
catalysis. A total binding site can include several subunits of an
oligomeric enzyme, as in the case of DNA-dependent
oligomeric polymerases.
Contrary to the total binding site of the enzyme, a subsite always
interacts with a polymer DNA within the context of a total binding
site. A subsite cannot be used as an independent entity in the
interactions of the enzyme with polymer DNA; nor can it independently
perform the catalysis.
The Total Binding Site of the DnaB Helicase Is Structurally
Heterogeneous--
Nuclease digestion protection studies provide a
clear indication of the structural heterogeneity of the total binding
site of the E. coli DnaB helicase. Only 10 or 11 nucleotide
residues, within the total binding site, are strongly protected from
digestion, while the remaining 9 or 10 residues are accessible to the
nuclease (Fig. 1, a and b). These results
indicate that the total binding site of the helicase, which occludes on
~20 nucleotide residues in the complex with polymer ssDNA, is built
of two binding subsites each encompassing a similar number of ~10
nucleotides. Experiments on the binding of partial DNA ligands to the
helicase showed a large difference in the affinities between the
subsites and indicated that the 5'-end of the nucleic acid interacts
with the strong binding subsite of the total binding site of the
enzyme. The fact that the nuclease can access ~half of the total
number of occluded residues within the entire binding site suggests not
only a difference in the affinities between the subsites but also an
open architecture of the hexamer at the subsite that encompasses the
3'-end of the nucleic acid (see below).
The Two DNA Binding Subsites of the Total Binding Site of the DnaB
Helicase Have Dramatically Different Affinities for ssDNA--
Direct
evidence of large differences in the affinities between the DNA binding
subsites of the DnaB helicase comes from the studies of the binding of
a partial ligand, d
A(p
A)9, to the enzyme. Using the
thermodynamically rigorous method, we determined that only one 10-mer
binds with significant affinity to the helicase and that the
association is characterized by the binding constant K1 = (1.7 ± 0.3) × 107
M
1. The affinity for the second binding
subsite is characterized by K2 = (2.6 ± 1) × 104 M
1; thus, it is ~3
orders of magnitude lower. It is evident that the major part of the
free energy of binding of the helicase to ssDNA comes from interactions
with the strong binding subsite. The very low affinity of the weak
binding subsite indicates that the protein does not form efficient
contacts with a single-stranded nucleic acid and suggests that this
subsite of the helicase is not functionally a ssDNA binding site but
rather that it fulfills a different role when the enzyme is in the
complex with its physiological substrate, the replication fork (see
below).
To efficiently form a complex with the strong DNA binding subsite, the
nucleic acid must have a length of at least 6 or 7 nucleotide residues.
No detectable affinities were observed with ssDNA oligomers shorter
than 6 nucleotides in our solution conditions (Fig. 4a). It
is interesting that the difference of 2 residues between 7- and 5-mer
practically abolishes the affinity of the shorter oligomer for the
binding site, while the same difference between the 8- and 10-mer leads
to a decrease of the free energy of binding by only ~0.3 kcal/mol
(Fig. 4b). A common misconception in studying
protein-nucleic acid interactions is treating both a nucleic acid and a
protein as interacting regular lattices. The difference between the
free energy of interaction of oligomers of different lengths with the
protein is then assigned to the difference in statistical effects
between different oligomers, which usually has very poor quantitative
justification. We point out that the nucleic acid is the only
macromolecule that can be approximated by a regular lattice. The
binding site on the protein can have a very complex structure, with
distant regions making key contacts with the nucleic acid, hardly
resembling a regular lattice. The differences between different
oligomers in binding to the DnaB helicase cannot be explained by any
difference in the statistical effect between the oligomers. This is
particularly true for oligomers shorter than 6 or 7 residues. Rather,
the results suggest that the elements of the strong binding subsite of
the enzyme, which makes crucial binding contacts with the nucleic acid,
are separated by a distance spanned by 6 or 7 nucleotides. The proper
complex is formed only when all essential contacts are engaged in
interactions with ssDNA. In this context, the similar number of ions
released in the interactions of a 10-mer and a 6-mer with the helicase
(~1.5) would be a result of the fact that a 6-mer can still form all
essential contacts with the enzyme, although the oligomer constitutes
only 60% of the length of the 10-mer (Fig. 5b).
Direct Proof That the DnaB Helicase Binds in a Single Orientation
with Respect to the Sugar-Phosphate Backbone of a ssDNA--
Most of
the studied helicases show preferential direction in the unwinding of
dsDNA, i.e. in the 5'
3' or the 3'
5' direction (30). Therefore, it is often a priori assumed that the
enzyme binds in strict polarity, 5'
3' or 3'
5', with respect
to the orientation of the single-stranded nucleic acid strand. This is
a natural assumption that simplifies current models, based on the still
limited solution data, of how the helicase functions at the replication
fork. However, one can argue that the enzyme can bind in both
orientations to the nucleic acid lattice and that the proper
orientation is imposed by specific interactions with dsDNA and/or
multiple proteins that are building the machinery of the replication
fork. In this context, it should be noted that several specific
protein-protein interactions between the DnaB helicase and proteins,
which are part of the primosome or replication fork complex, have been
identified. Although recent electron microscopy and crystallographic
data show polarity in a helicase binding to ssDNA (31, 32), the
polarity in the binding of a helicase, with respect to the
directionality of a ssDNA strand, has never been directly shown for any
hexameric helicase in solution.
As we pointed out, the determination of the mutual orientation of the
protein and nucleic acid in a complex should be based on the method
that is sensitive to the differences in distances between different
specific regions of both macromolecules. The fluorescence energy
transfer technique is such a method. The difference in the effect of
the location of the acceptor, fluorescein, at the 5'- or 3'-end of the
20-mer, dT(pT)19, on the fluorescence spectra of the
complex of nucleic acid with R14C-CPM (excited in a predominantly donor
absorption band), is dramatic (Figs. 6 and 7). These dramatic spectral
differences are reflected in the large differences between the energy
transfer efficiencies from CPM in the small 12-kDa domains, all located
at one end of the DnaB hexamer, and the fluorescein placed at the 5'-
or 3'-end of the dT(pT)19, which spans the entire DNA
binding site. The efficiency, E, for the fluorescein placed
at the 5'-end of the 20-mer is 0.71 ± 0.04. The efficiency of the
same acceptor located at the 3'-end of the nucleic acid is only
0.10 ± 0.01. In the case of chemically identical donor-acceptor
pairs, the transfer efficiency depends on two variable factors
characteristic for the studied system, the distance between the donor
and acceptor, R, and the orientation parameter,
2, which characterizes the mutual orientation of the
donor absorption dipole and acceptor emission dipole (22). The value of
2 can theoretically assume any value between 0 and 4, but only these two extreme values would significantly affect the
determined transfer efficiency. The possible range of
2
can be estimated by using the standard procedure (Ref. 24; Table II).
The obtained ranges of
2 are very similar for both
5'-Fl-dT(pT)19 and dT(pT)19-Fl-3' and away from
the extreme values of 0 and 4 (Table II). Another equally rigorous
procedure is to perform experiments with several different donor-acceptor pairs that, due to different structures of different chromophores, provide the necessary "randomization" of the
orientations of emission and absorption dipoles (33). Using different
donor-acceptor pairs, we obtained similar, very large differences
between the fluorescence energy transfer efficiencies from the
fluorophore on the 12-kDa domain of the DnaB helicase and the donor or
acceptor placed at the 5'- and the 3'-end of the bound 20-mer (data not shown). The results clearly show that the large difference between the
transfer efficiencies results from the large difference in the
distances between the 5'-end and the 3'-end of the 20-mer and the CPM
located on the small domain of the DnaB protomers.
Our data show that the DnaB helicase binds ssDNA in a predominately
single orientation, with respect to the polarity of the single-stranded
nucleic acid lattice. Moreover, the data show that, in the complex with
ssDNA, the small domain of the protein is in close proximity to the
5'-end of the nucleic acid, while the large domain is located near the
3'-end of the bound ssDNA.
Sequential Locations of the Strong and Weak Binding Subsites of the
DnaB Helicase--
As determined in this work, the partial ligand,
d
A(p
A)9, binds with overwhelming preference to the
strong binding subsite of the DnaB helicase. The transfer efficiency
between the 5'-end of the 10-mer, dA(pA)9, labeled with
fluorescein, and CPM, located in the small domain of the DnaB protein,
is very similar to the transfer efficiency between the CPM and
fluorescein at the 5'-end of the 20-mer, dT(pT)19 (data not
shown). These results indicate that the 5'-ends of both the 10- and
20-mers are at a similar distance from the small domain of the protein.
Thus, the strong ssDNA binding subsite encompasses the bound 20-mer at
its 5'-end, which is in close proximity to the small 12-kDa domain of
the protein, while the weak binding subsite is located entirely on the
large domain. At present, it is unknown whether or not the small domain
or the hinge region of the DnaB protomer are directly involved in
interactions with ssDNA. It should be noted that the isolated large
33-kDa domain of the enzyme could still bind ssDNA with some affinity,
although quantitative analysis of the binding has not been performed
(28). Thus, it is possible that the small domain and the hinge region
constitute a part of the strong ssDNA binding subsite of the intact
DnaB helicase.
The DnaB helicase binds preferentially to the 5'-arm of the replication
fork (26). Because the enzyme binds in a single orientation, with
respect to the polarity of the ssDNA sugar-phosphate backbone, with the
small domain facing the 5'-end of the ssDNA, it is evident that in the
complex with the replication fork, the helicase hexamer is oriented
with the large domains of the protomers toward the duplex part of the
fork, while the 5'-end of the arm of the replication fork is located in
the vicinity of the small 12-kDa domains of the protomers. Anisotropy
of the probe located at the 5'-end of the 20-mer bound to the helicase
is significantly higher than the anisotropy of the same fluorescein
residue located at the 3'-end of the nucleic acid (Table II). A
significant decrease of the anisotropy, when the probe is located at
the 3'-end of the bound nucleic acid, indicates an increased mobility
of the nucleic acid in the weak binding subsite and is most probably due to the lack of strong contacts between the single-stranded nucleic
acid and the binding site. Recall that the micrococcal nuclease can
access the part of the nucleic acid in the weak subsite of the total
binding site of the DnaB helicase, suggesting a more open structure of
the total binding site at the 3'-end of the bound 20-mer.
The results described in this work provide an insight into the complex
structure-function relationship within the DNA binding site of a
replicative hexameric helicase. A model of the single, total DNA
binding site on the DnaB protomer, engaged in the complex with the
replication fork and based on the data presented in this work, is
schematically shown in Fig. 8. The total
DNA binding site of the enzyme is built of two subsites placed
sequentially along the DNA substrate in the protein-nucleic
acid complex. The strong ssDNA binding subsite occludes the 5'-end of
the ssDNA, is located in close proximity to the small 12-kDa domain,
and is distant from the duplex part of the fork. Binding of ssDNA to
this subsite leads to the significant immobilization of the nucleic
acid and provides the major part of the binding free energy. The
subsite, which is located at the 3'-end of the ssDNA, binds the
single-stranded nucleic acid very weakly. The single orientation of the
helicase in the complex with ssDNA indicates that, when the enzyme
approaches the replication fork, it faces the duplex part of the fork
with the weak binding subsite located entirely on the large 33-kDa
domain of the protein. Thus, the weak binding subsite constitutes the
entry site for the dsDNA in the fork. The more open architecture of
this subsite provides a large space, which is necessary for the
incoming duplex DNA.

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Fig. 8.
Schematic representation of the mutual
orientation of the small 12-kDa and large 33-kDa domains of the single
DnaB helicase protomer and the DNA binding subsites with respect to the
polarity of the ssDNA, arms, and duplex DNA in the complex of the
enzyme with replication fork, based on the results obtained in this
work. The helicase is preferentially bound to the 5'-arm of the
replication fork using a single, total binding site of one of the six
protomers shown in the figure. The locations of the DNA binding
subsites within the total binding site is sequential. The
weak binding subsite on the large 33-kDa domain faces the duplex part
of the fork and constitutes the entry site for the dsDNA. The strong
binding subsite is in the vicinity of the small 12-kDa domain and is
engaged in interactions with the ssDNA at the 5'-end of the arm. The
3'-arm is not forming a stable complex with the helicase hexamer
associated with the 5'-arm of the fork (26).
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Comparison with other hexameric helicases is difficult because, at this
time, no analogous data on the structure of their nucleic acid binding
sites are available. However, it is possible that similar functional
and structural relationships within the DNA binding site are general
for all other hexameric helicases.
We thank Dr. T. Wood from the NIEHS Center,
National Institutes of Health, for excellent work in obtaining the DnaB
protein variant R14C and for many helpful discussions. We thank Gloria Drennan Davis for help in preparing the manuscript.