Hepatitis C virus (HCV) helicase catalyzes
the ATP-dependent strand separation of duplex RNA and DNA
containing a 3' single-stranded tail. Equilibrium and velocity
sedimentation centrifugation experiments demonstrated that the
enzyme was monomeric in the presence of DNA and ATP analogues.
Steady-state and pre-steady-state kinetics for helicase activity were
monitored by the fluorescence changes associated with strand separation
of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged
31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21).
kcat for this reaction was 0.12 s
1. The fluorescence change associated with strand
separation of F21:HF31 by excess enzyme and ATP was a biphasic process.
The time course of the early phase (duplex unwinding) suggested only a
few base pairs (~2) were disrupted concertedly. The maximal value of
the rate constant (keff) describing the late
phase of the reaction (strand separation) was 0.5 s
1,
which was 4-fold greater than kcat. Release of
HF31 from E·HF31 in the presence of ATP (0.21 s
1) was the major contributor to
kcat. At saturating ATP and competitor DNA
concentrations, the enzyme unwound 44% of F21:HF31 that was initially
bound to the enzyme (low processivity). These results are consistent
with a passive mechanism for strand separation of F21:HF31 by HCV
helicase.
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INTRODUCTION |
Helicases are ubiquitous enzymes required for cellular repair,
recombination, and replication (1, 2). Even though helicase activity
was identified and the associated protein was purified over 20 years
ago (3), the kinetic and chemical mechanism for this class of enzymes
is unknown. Lohman and co-workers (4-13) have initiated an extensive
effort to elucidate the mechanism of action of Escherichia
coli Rep helicase. They have proposed that the catalytically
active species is a dimeric form of the enzyme stabilized by DNA (2, 4,
12). The two DNA-binding sites in the catalytically active dimer unwind
defined lengths of the DNA duplex by alternatively binding duplex and
single-stranded DNA during the catalytic cycle (2, 6, 7, 13). Recently, Ali and Lohman (14) reported that E. coli helicase II
catalyzes the unwinding of defined duplexes with a step size of 4 to 5 base pairs per catalytic cycle. These results supported an active
mechanism for separating double-stranded DNA in which the step size is
relatively small.
Our interest in helicases is based on the observation that the
HCV1 genome encodes a unique
helicase within the NS3 protein. Because approximately 1% of the
population is infected with HCV and available therapies are effective
for only a small subpopulation of these patients, an urgent medical
need exists for an effective anti-HCV agent (15-17). Consequently, HCV
helicase is an attractive target for development of an antiviral agent
for HCV.
The NS3 protein of HCV has at least two enzymatic activities necessary
for viral replication. The N-terminal 20 kDa of NS3 is a serine
proteinase that cleaves the HCV-encoded polyprotein at four specific
positions (18). The C-terminal 50 kDa of NS3 has NTPase (19) and RNA
helicase activity (20). We have initiated a program to characterize the
kinetic and chemical mechanism of action for the HCV helicase domain.
The kinetic mechanism of the ATPase activity associated with this
protein isolated from HCV genotype 1b, which is a major subtype found
in both Japanese and American populations (21), has been characterized
in detail (22). Herein, we extend our understanding of the kinetic
mechanism of the helicase activity of this protein by analysis of the
strand separation reaction with fluorescently tagged duplex oligomers of defined sequences. The results with HCV helicase were consistent with a passive kinetic mechanism for DNA strand separation in which
dissociation of single-stranded DNA was the major contributor to
kcat.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Rabbit muscle pyruvate kinase, rabbit muscle
lactate dehydrogenase, NADH, phosphoenolpyruvate, adenosine
5'-triphosphate, AMPCPP, ATP
S, ADPPNP, MOPS, Tris, ammonium sulfate,
and dextran sulfate (Mr = 5000) were from Sigma.
ATP, poly(rU), and single-stranded DNA cellulose resin were from
Amersham Pharmacia Biotech. All oligomers of defined sequence were
purchased from Oligo Therapeutics, Inc., and were purified by
electrophoresis through a 6 M urea, 20% polyacrylamide
gel.
Purification of the HCV Helicase--
The enzyme was purified as
described previously except that a single-stranded DNA cellulose column
was substituted for the Bio-Gel P-100 column in the previous
purification protocol (22). In summary, the enzymatic activity from the
Mono Q purification step (22) was absorbed onto a column of the
single-stranded DNA-cellulose resin (10 g) that was equilibrated in the
purification buffer. The flow rate was 1 ml/min. After washing the
resin with 10 ml of buffer containing 0.15 M NaCl, the
protein was eluted from the resin with buffer containing 1.0 M NaCl. The purified enzyme was stored at
20 °C for
several months without loss of catalytic activity.
Assay of HCV Helicase Activities--
The ATPase activity
associated with HCV helicase was monitored by the
ADP-dependent oxidation of NADH in the lactate
dehydrogenase and pyruvate kinase-coupled reaction as described
previously (22).
Several continuous read-out fluorescence-based assays have been
recently developed for the study of DNA strand separation catalyzed by
helicases (10, 13, 23-27). For the HCV helicase reaction, we chose the
fluorescence resonance energy assay developed by Bjornson et
al. (10). The substrate was a double-stranded DNA of fluorescein-
and hexachlorofluorescein-tagged oligomers (10). The fluorescence of
the fluorescein moiety (
ex = 492 nm and
em = 522 nm) in the double-stranded DNA was quenched by fluorescence resonance energy transfer to the hexachlorofluorescein moiety (10). Separation of the fluorescein and hexachlorofluorescein moieties results in an increase in fluorescence of the fluorescein moiety (10). For our studies, a 21-mer and a complementary 31-mer were
modified with fluorescein and hexachlorofluorescein on the 3' end (F21)
and 5' end (HF31), respectively. The fluorescence quenching we observed
upon formation of F21:HF31 was approximately 2-fold greater than that
observed by Bjornson et al. (10) for the formation of an
analogously tagged duplex DNA. The fluorescence changes associated with
strand separation of F21:HF31 by sub-stoichiometric concentrations of
enzyme were proportional to the fractional strand separation of
F21:HF31 determined by a gel-based assay (data not shown).
Kinetic and Titration Data Analyses--
Equation 1, which
describes a random rapid equilibrium or steady-state ordered mechanism
(28), was fitted to the steady-state data to determine the value of
kcat for strand separation of F21:HF31.
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(Eq. 1)
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KATP, F21:HF31 is the
Km of the enzyme for F21:HF31 extrapolated to
infinite ATP concentration; KF21:HF31,ATP is the
Km of the enzyme for ATP extrapolated to infinite F21:HF31 concentration; and KATP is the
Kd of the enzyme for ATP in the absence of F21:HF31.
The time courses for changes in the intrinsic protein fluorescence
resulting from binding of DNA to helicase were described by either a
single (n = 1) or double (n = 2)
exponential function (Equation 2).
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(Eq. 2)
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The dependence of the kobs values on DNA
concentration ([L]) was described by either a two-step mechanism or a
one-step mechanism (Equation 3 or 4).
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(Eq. 3)
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(Eq. 4)
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Equations 5 and 6 were fitted to the biphasic fluorescence
changes associated with HCV-catalyzed strand separation of F21:HF31, 21:HF31, and 42:HF52 in half-reactions or partial turnover experiments. The early phase was a linear change in fluorescence from an initial value (c) to a final value (d) over a time
interval (a) (Equation 5). The late phase was an exponential
change in fluorescence from an initial value (d) to the
final fluorescence (e) (Equation 6).
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(Eq. 5)
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(Eq. 6)
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Sedimentation Velocity Centrifugation--
Sedimentation
velocity analytical ultracentrifugation was performed with a Beckman
(Fullerton, CA) XL-A centrifuge with two-channel 12-mm charcoal-filled
epon centerpieces. Scans were taken at 280 nm for HCV helicase alone or
at 492 nm for experiments with 3' fluorescein-tagged DNA
(I-F) at 10-min intervals over a 5-h period. Centrifugation
was at 40,000 rpm at 4 °C, except in the case of I-F
alone, which was performed at 60,000 rpm. Each experimental solution
(200 µl) was centrifuged against 250 µl of the equivalent blank.
The apparent sedimentation coefficient, Sapp,
was calculated from the apparent differential sedimentation coefficient
distribution, using a software analysis package created by Stafford
(29) that was obtained through the National Analytical Ultracentrifugation Facility at the University of Connecticut (Storrs,
CT).
Sedimentation Equilibrium Centrifugation--
Sedimentation
equilibrium analytical ultracentrifugation was performed as described
above except that six-sectored cells were utilized. Scans were taken at
280 nm at 1-h intervals throughout the run. Runs were performed at
20,000, 25,000, and 30,000 rpm at 4 °C. Equilibrium was achieved
after approximately 20 h. Each sample (80-100 µl) was
centrifuged against 120 µl of the equivalent blank. Solvent density
was determined empirically at 4 °C using a Mettler (Highstown, NJ)
DA-110 density/specific gravity meter calibrated against water. The
partial specific volume of each protein,
, was calculated
using the method of Cohn and Edsall (30). Adjustments for temperature
were made using the appropriate equation that has been modified to use
values derived for each amino acid at 25 °C (31). The
partial specific volume of HCV helicase was calculated to be 0.726 ml/g
at 4 °C. The value of
for I was 0.55 ml/g. Data
were collected as radial distance versus absorbance. The
data were analyzed by an adaptation of the Beckman/Microcal Origin
nonlinear regression software package using multiple iterations of the
Marquardt-Levenberg algorithm for parameter estimation. Multiple models
were employed to determine the most accurate description of the
macromolecular solution state.
|
(Eq. 7)
|
The sedimentation of a single, ideal species was described by
the first term on the right-hand side of Equation 7.
Ar was the absorbance at radial position
r. A01 was the absorbance of the
single species monomer at r0 (the reference
radial position at the top of the gradient).
1 was
the partial specific volume;
was the solvent density;
was the
angular velocity; R was the gas constant; T was
the temperature in degrees Kelvin; and M1 was
the molecular mass. The base-line offset was E. This
analysis yielded an approximate value for the mass average molecular
mass of the sedimenting species in the system. This single ideal
species model was sufficient to describe the sedimentation of helicase or DNA alone. The description of the sedimentation of helicase in the
presence of DNA required all the terms of Equation 7, which included
terms for each of the individual sedimenting monomeric species, the
hetero-complex species, and a base-line offset. The variables were
defined as for the single, ideal species model. The variables
subscripted with 1 and 2 refer to parameters for helicase and DNA,
respectively. In addition, n1 and
n2 referred to the number of molecules of
helicase and DNA, respectively, that were in the hetero-complex.
General Methods--
The standard buffer was 0.05 M
MOPS (K+), 3.5 mM MgCl2 at pH 7.0. The standard temperature was 25 °C. Duplex DNA was made from stoichiometric concentrations of single-stranded DNA by heating the
solution to 90 °C for 3 min and then cooling to room temperature over several hours. Fluorescence spectra were recorded on a Kontron SFM
25 spectrofluorometer. Intrinsic protein fluorescence data (
ex = 280-300 nm and
em = 340 nm) were
corrected for filter effects. F21 was monitored with
ex = 492 nm and
em = 522 nm; HF31 was monitored with
ex = 510 nm and
em = 552 nm with a 2-mm slit. Absorbance and conventional kinetic data were obtained from a
UVIKON 860 spectrophotometer. Rapid reactions were monitored on an
Applied Photophysics SX.17MV Spectrophotometer (Leatherhead, UK).
Entrance and exit slits were 10 mm in the absorbance mode and 2 mm in
the fluorescence mode. The fluorescence of F21 and HF31 was monitored
on the stopped-flow spectrofluorometer with
ex = 492 nm
and
ex >530. The intrinsic protein fluorescence was
monitored with
ex between 280 and 290 nm and
ex >305 or >320 nm. Stopped-flow tracings were an
average of 4-6 experiments. The appropriate equations were fitted to
the data by nonlinear least squares using SigmaPlot from Jandel
Scientific (Corte Madera, CA).
 |
RESULTS |
Nucleotide Triphosphates and DNA Did Not Induce Oligomerization of
HCV Helicase--
HCV helicase is isolated as a monomeric protein
(22). Many helicases that are monomeric in the absence of substrates
undergo substrate-induced oligomerization to the catalytically active form (32, 33). This frequently results in a nonlinear dependence of the
rate of the reaction on enzyme concentration. For instance, the ATPase
activity of E. coli Rep helicase, which undergoes
substrate-induced dimerization, is a nonlinear function of enzyme
concentration (2, 12). The ATPase activity of HCV helicase, however,
was linearly dependent on helicase concentration between 0 to 50 nM enzyme with 1000 µM ATP and 600 µM poly(rU) (monomer). Similarly, the DNA unwinding
activity was linearly dependent on helicase concentration over a range
of 0 to 25 nM enzyme with 2000 µM ATP and 17 nM F21:HF31 as substrates. These results suggested that either the oligomerization of HCV helicase did not change over this
range of enzyme concentrations or changes in oligomerization of
the enzyme did not affect catalysis.
The oligomerization of HCV helicase in the presence of substrate
analogues was investigated further by sedimentation equilibrium centrifugation. A duplex DNA with a stem-loop structure (GGC CTA AGC
GTA TCG CTT AGG CCG AGT CAG G, I) was chosen for these studies because 1) it bound the helicase tightly with a 1:1
stoichiometry, 2) it mimicked double-stranded DNA and RNA that HCV
helicase normally unwinds (34, 35), and 3) if unwound by the enzyme, it
would reanneal rapidly. Sedimentation equilibrium data for HCV helicase alone or I alone (4 to 20 µM) were described
by the first term of Equation 7 that described sedimentation of a
single ideal species. The fitted values for the molecular mass of
enzyme and I were 49,000 ± 1000 and 12,000 ± 1000 Da, respectively, which were within error of that expected for the
respective monomeric macromolecules.
Solutions of mixtures of helicase and I exhibited more
complicated sedimentation behavior. A model that includes one hetero-associating species was found to adequately fit the experimental results (Equation 7). Goodness of the fit was judged by several criteria as follows: 1) the value of the reduced sums of the squares of
the residuals, 2) a random distribution pattern of the residuals to the
fit, and 3) the appropriate nature of the model to the system being
studied. Fig. 1 represents an example of
the best fit to this model. The random distribution of residuals is
shown above the data. The apparent dissociation constant for the 1:1 hetero-complex was less than 1 µM.2 Species
distribution analysis using this association constant indicated that
greater than 70% of the macromolecules were in this hetero-complex
under these experimental conditions. Attempts to fit models to the data
having hetero-species with other than 1:1 stoichiometry resulted in a
less random distribution of residuals and values for association
constants that translated to a very small (less than 5%)
hetero-species population.

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Fig. 1.
Equilibrium analytical ultracentrifugation of
HCV helicase·I. A mixture of 10 µM helicase and 10 µM I were centrifuged at 20,000 RPM and
4 °C in the standard buffer. Data are represented as absorbance at
280 nm versus radial position. Sedimentation gradients were
analyzed by nonlinear curve fitting using equations describing a
heterogeneous molecular association model, as described under
"Experimental Procedures." The solid line through the
data points was the best fit to the data for a 1:1 complex between
helicase and I. Residuals to the best fit to the data are
shown above the data.
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Because the HVC helicase has significant ATPase activity, it was not
experimentally feasible to determine the effect of ATP on the
oligomerization of the enzyme by sedimentation equilibrium centrifugation. Consequently, the effects of I and ATP analogues on the oligomerization of HCV helicase were investigated by
sedimentation velocity centrifugation. In these experiments, I tagged on the 3' end with a fluorescein moiety
(I-F) allowed sedimentation of E·I-F
to be monitored at wavelengths (
max = 492 nm) without
influence from nucleotides (
max = 260 nm). The
Sapp values for E·I-F in
the presence or absence of ATP analogues were similar (Table
I). If the substrates induced dimerization of the helicase, the sedimentation coefficient would have
been expected to increase by 50% assuming helicase was accurately described by a spherical molecule (32). Thus, the sedimentation velocity and equilibrium data suggested that
I·F or the combination of
I·F and ATP analogues did not affect
oligomerization of this protein.
Steady-state Kinetics for Unwinding of HF31:F21 by HCV Helicase and
ATP--
Initial velocity data for unwinding F21:HF31 by HCV helicase
([E]
[F21:HF31]) and ATP were monitored as a
fluorescence increase associated with enzymatic separation of the
duplex (Fig. 2). These data were analyzed
by Equation 1. The value of kcat was 0.123 ± 0.009 s
1. The values of the other parameters were
KATP, F21:HF31 = 0.030 ± 0.006 µM, KF21:HF31, ATP = 360 ± 90 µM, and KATP = 1600 ± 400 µM.

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Fig. 2.
Steady-state velocities for unwinding of
F21:HF31 by helicase as a function of ATP and F21:HF31
concentrations. Strand separation of F21:HF 31 was monitored as
described under "Experimental Procedures." The reactions were
initiated with 1.1 nM helicase. The slope of the time
course for the fluorescence change was calculated over approximately
100 s. Equation 1 was fitted to the data to give values for
kcat = 0.124 s 1,
KATP, F21:HF31 = 0.030 µM,
KF21:HF31,ATP = 360 µM, and
KATP = 1600 µM. The solid
lines were calculated with these fitted values and Equation 1.
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Reaction of E·ATP with F21:HF31 Monitoring Enzyme
Fluorescence--
The reaction of E·ATP (because of the
ATPase activity of the enzyme this species was a possible mixture of
E·ATP, E·ADP·Pi, and
E·ADP and E·Pi) with excess
F21:HF31 and ATP (Fig. 3) resulted in a
time-dependent decrease in intrinsic protein fluorescence resulting from formation of E·ATP·F21:HF31 (Equation 8).
Because ATP and F21:HF31 were in excess of enzyme, the fluorescence
changes were monitoring the approach of the system to the steady
state.

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Fig. 3.
Kinetics for the approach to the steady-state
starting with E·ATP and F21:HF31. The reaction of
E·ATP with F21:HF31 was monitored by the change in
intrinsic protein fluorescence ( ex = 280 nm and
em >305 nm) associated with the formation of
E·ATP·F21:HF31 at large concentrations of ATP
(inset). The time course for the reaction of 50 nM E·ATP with 400 nM F21:HF31 in
the presence of 1000 µM ATP was first-order in enzyme
concentration (inset). Equation 2 (i = 1)
was fitted to these data to give a pseudo first-order rate constant of
2.4 s 1. The value of kobs for the
reaction of 240 nM F21:HF31 was similar with 500 and 2000 µM ATP. The value of the bimolecular rate constant for
association of E·ATP with F21:HF31 was 3.4 µM 1 s 1
(k1,ATP in Equation 9). The intercept value was
0.96 s 1.
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|
The pseudo first-order rate constant for the approach to the
steady-state (kobs) for the simplified scheme of
Equation 8 was given by Equation 9. The values of
kobs were linearly dependent on F21:HF31
concentration (Fig. 3) and similar for E·ATP formed with
500, 1000, and 2000 µM ATP (data not shown).
|
(Eq. 9)
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The values calculated for k1,ATP and the
sum of kcat and k
1,ATP
were 3.4 ± 0.1 µM
1 s
1
and 0.96 ± 0.09 s
1, respectively (Equation 9).
Thus, the calculated value of k
1,ATP was 0.84 s
1 (kcat = 0.12 s
1).
The value of k1,ATP was similar to the value of
kcat/kATP, F21:HF31 (4.1 µM
1 s
1) from steady-state
data indicating that the pre-steady-state data for the reaction of
E·ATP with F21:HF31 was measuring an event associated with
catalysis.
Half-reaction of E·F21:HF31 with ATP: Processivity of
E·F21:HF31--
The fraction of F21:HF31 in E·F21:HF31 that was
separated into F21 and HF31 (separation of fluorescent probes) after
addition of ATP in the presence or absence of excess single-stranded
competitor was estimated from the increase in fluorescence of
fluorescein (
ex = 492 nm and
em = 522 nm). E·F21:HF31 (50 nM) was formed from
stoichiometric amounts of enzyme and F21:HF31 in the absence of ATP
(Kd = 0.4 nM). In the absence of
competitor (dU)18, the increase in fluorescence of F21
after addition of 2000 µM ATP was the result of total
strand separation (inset Fig.
4 with addition of the mixture of ATP and
(dU)18 at the time indicated by the arrow). In
the presence of competitor (dU)18, only a fraction of
F21:HF31 strands was separated. This result suggested that a
significant fraction of F21:HF31 or partially unwound F21:HF31 in
E·F21:HF31·ATP dissociated from the enzyme prior to
strand separation (i.e. the processivity of the enzyme was
low). The ratio of the fluorescence changes in the presence
(dU)18 to that in the absence was dependent on ATP
concentration. The maximal fractional strand separation extrapolated to
infinite ATP concentration was 0.44 ± 0.03. The concentration of
ATP that yielded 50% of the maximal fractional strand separation was
1300 ± 200 µM (Fig. 4). The fractional strand
separation was not affected by enzyme concentration (200 nM
enzyme, 50 nM F21:HF31), which suggested that multiple
enzyme molecules were not binding to a single molecule of F21:HF31.

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Fig. 4.
Dependence of fractional strand separation of
F21:HF31 in E·F21:HF31 on ATP concentration in the
presence of competitor DNA. The formation of F21 from F21:HF31 in
the presence of ATP and HCV helicase was monitored by the fluorescence
increase at 522 nm ( ex = 492 nm).
E·F21:HF31 was formed from 50 nM F21:HF31 and
58 nM E. The reaction was initiated by the
simultaneous addition of selected concentrations of ATP and 4.0 µM (dU)18 to trap free enzyme as F21:HF31 or
strand separated DNA was released from E·F21:HF31·ATP.
An example of the time course for the reaction with 2000 µM ATP in the absence of (dU)18 (total strand
separation) or in the presence of 4.0 µM
(dU)18 (partial strand separation) is presented in the
inset. At the time indicated by the arrow the
mixture of ATP and (dU)18 was added simultaneously to
E·F21:HF31. The fractional strand separation (ratio of the
fluorescence change in the presence of (dU)18 to that in
the absence of (dU)18) was depended on the ATP
concentration. The maximal fractional strand separation in the presence
of ATP and (dU)18 was 0.44 and the concentration of ATP
yielding 50% of this value was 1300 µM.
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Half-reaction of E·F21:HF31 with ATP: Kinetics of Strand
Unwinding and Strand Separation--
The half-reaction of 50 nM E·F21:HF31 with 2000 µM ATP
in the presence of 40 µM competitor DNA
((dU)18), which was added simultaneously with ATP to
E·F21:HF31, resulted in a biphasic fluorescence increase (
ex = 492 nm and
em >530 nm, Fig.
5). Under these conditions E·F21:HF31 was converted to 30% enzyme-free F21, 30%
enzyme-free HF31, and 70% F21:HF31. Equations 5 and 6 were fitted to
these data with a = 0.78 ± 0.01 s,
b = 0.26 ± 0.01 s
1,
c = 0 (fixed), d =
0.001 ± 0.0002, and e = 1 (fixed). Because the observation
wavelengths were monitoring fluorescence changes associated with
formation of F21 and dissociation of E·HF31 (see below),
the kinetics of the late phase of the reaction were complicated. Nonetheless, the time course for the reaction demonstrated a distinct lag (0.78 s) prior to formation of enzyme-free F21 and HF31. The early
phase was attributed to unwinding of the duplex prior to complete
strand separation. Because the fluorescent probes were not separated
during this phase, the fluorescent change associated with this reaction
was small. The late phase was attributed to strand separation. Because
the fluorescent probes were separated in this phase, the fluorescent
changes were large (see "Discussion").

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Fig. 5.
Time course for the reaction of
E·F21:HF31 with ATP in the presence of competitor
DNA. 50 nM E·F21:HF31 was reacted with 2000 µM ATP in the presence of 4.0 µM competitor
(dU)18. The formation of F21 and HF31 was monitored with
the stopped-flow spectrofluorometer with ex = 492 nm and
em >530 nm. Equations 5 and 6 were fitted to the data
to give the following parameter values: a = 0.78 s, b = 0.26 s 1, c = 0 (fixed), d = 0.001, and e = 1 (fixed). The early phase of the reaction was attributed to unwinding
the duplex DNA. The late phase of the reaction was attributed to
complete strand separation with separation of the fluorescent
probes.
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Kinetics for Complete Strand Separation of F21:HF31 with [E]
[F21:HF31]--
To eliminate the contributions to the
fluorescence signal due to dissociation of E·HF31, the
reaction of F21:HF31 with E and ATP was repeated in the
absence of competitor DNA and with the enzyme concentration (>300
nM) much greater than the Km of
E·ATP for HF31 (30 nM). At the end of this
reaction the strands of F21:HF31 were completely separated, and HF31
was bound to the enzyme. A typical time course for the fluorescence
increase resulting from formation of F21 catalyzed by 348 nM helicase with 1000 µM ATP and 12.5 nM F21:HF31 is presented in the inset of Fig.
6. The fluorescence of the solution did
not increase significantly until 1 s after mixing the components.
This lag was not highly dependent on the order of addition of enzyme,
ATP and F21:HF31. After the lag phase, the fluorescence increased in a
first-order process. Equations 5 and 6 were fitted to these data to
give a lag time of 1.14 ± 0.05 s and a first-order rate
constant of 0.255 ± 0.002 s
1 (Fig. 6). The length
of the lag phase was independent of enzyme concentration, whereas the
value of the pseudo first-order rate constant for the late phase of the
reaction (kobs) was highly dependent on enzyme
concentration. The value of kobs for saturating helicase concentration with 2000 µM ATP was 0.50 ± 0.03 s
1. This was the maximal value for the effective
rate constant for strand separation at infinite enzyme concentration
(keff). The concentration of enzyme that gave
50% of this value was 360 ± 60 nM. With enzyme
and F21:HF31 concentrations fixed at 200 and 12.5 nM,
respectively, the dependence of kobs on ATP
concentration was described by Equation 3 (k
1 = 0) with k1 = 0.217 ± 0.008 s
1 and K = 350 ± 40 µM.

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Fig. 6.
Reaction of E·F21:HF31 with HCV
helicase and ATP in the absence of competitor (dU)18.
The time course for the reaction of 348 nM helicase with
1000 µM ATP and 12.5 nM F21:HF31 is given in
the inset. Equations 5 and 6 were fitted to the data to give
the following values for the parameters: a = 1.14 s, b = 0.255 s 1, c = 0 (fixed), d = 0.2, and e = 1 (fixed).
These values were used to calculate the solid line. The
pseudo first-order rate constant for the late phase of the reaction
with 2000 µM ATP was determined as a function of enzyme
concentration. Equation 3 (k 1 = 0) was fitted
to these data with k1 = 0.50 s 1and
K = 360 nM.
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Early Phase of the Reaction of E·ATP with 21:HF31 or 42:HF52 Was
Unwinding of the Duplex--
Fluorescence changes were also observed
(
ex = 492 nm and
em >530 nm) during the
reaction of E·ATP with 21:HF31. Because this substrate lacked the
fluorescein moiety on the 21-mer, these fluorescence changes were the
result of changes in the environment of HF31 during the unwinding
reaction. Experiments with free HF31 demonstrated that
E·ATP quenched the fluorescence of HF31 (
ex = 510 nm and
em = 552 nm) by 55%. In contrast to the
results observed with HF31, the fluorescence of HF31 in duplex 21:HF31 was not affected significantly by binding to E·ATP.
Consequently, the unwinding of 25 nM 21:HF31 in the
presence of excess enzyme ([E] > Km of
E·ATP for HF31) was predicted to quench the fluorescence
of HF31 in 21:HF31 only after separation of HF31 from 21 (Fig.
7). The enzyme initially formed
E·21:HF31 with little fluorescence quenching.
Subsequently, there was a small decrease in HF31 fluorescence that was
linearly dependent on time. The late phase of the reaction was a
first-order large decrease (~30%) in HF31 fluorescence.
Equations 5 and 6 were fitted to the data collected in the
presence of 1000 µM ATP to yield a = 1.78 ± 0.03 s, b = 0.427 ± 0.005 s
1, c = 1 (fixed), d = 0.865 ± 0.009, and e = 0 (fixed). The early phase
of this reaction was attributed to duplex unwinding, whereas the late
phase represented strand separation with concomitant isomerization of
E·HF31 to a less fluorescent complex.

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Fig. 7.
Effect of the length of duplex DNA on the lag
in product formation. Time course for the reaction of 475 nM helicase with 25 nM 21:HF31 (left
tracing) or 25 nM 42:HF52 (right tracing)
in the presence of 1000 µM ATP. Fractional fluorescence
changes were normalized to the total fluorescence change observed for
each reaction (33 and 30% fluorescence quenching for 21:HF31 and
42:HF52, respectively). Equations 5 and 6 were fitted to the data for
the reaction of 21:HF31 with the enzyme to give the following values
for the parameters: a = 1.78 s, b = 0.427 s 1, c = 1 (fixed),
d = 0.865, and e = 0 (fixed). The
values for the parameters for the reaction of 42:HF52 with the enzyme
were a = 4.32 s, b = 0.119 s 1, c = 1 (fixed), d = 0.898, and e = 0 (fixed).
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This interpretation was verified by demonstrating that the lag time
(a) was related to the length of the duplex substrate. In
particular, the lag time determined for unwinding 42:HF52 was approximately twice as long as that for 21:HF31 (Fig. 7). The 42-mer
was a tandem duplication of the sequence of the 21-mer, and the
complementary 52-mer was analogous to HF31. Equations 5 and 6 were
fitted to the data for the reaction of 25 nM 42:HF52 with
475 nM helicase (Fig. 7) to yield a = 4.32 ± 0.05 s, b = 0.1195 ± 0.0006 s
1, c = 1 (fixed), d = 0.898 ± 0.005, and e = 0 (fixed). The lag phase was 2.4-fold longer with the 2-fold longer duplex, and the value
of the pseudo first-order rate constant was 25% that for the shorter
double-stranded DNA. Thus, the direct correlation between the length of
the double-stranded DNA and the length of the early phase supported the
interpretation that continuous unwinding of double-stranded DNA
occurred in the early phase of the reaction.
Dissociation of F21 and HF31 from E-DNA Complexes--
The value
of kcat for strand separation of F21:HF31 by HCV
helicase was 0.12 s
1, which was significantly less that
the maximal value for the effective rate constant for strand separation
in a half-reaction of E·F21:HF31 with ATP (0.5 s
1, Fig. 6). Consequently, another kinetic step such as
dissociation of product (F21 or HF31) from the enzyme-DNA complex was a
major contributor to kcat. This possibility was
also suggested from differences in the time course for formation of
enzyme-free F21 and enzyme-free HF31 (Fig.
8A). In these experiments,
E·F21:HF31 was reacted with 2000 µM ATP and
200 µg/ml poly(rU). F21 was monitored by the fluorescence increase
with
ex = 492 nm and
em = 522 nm. HF31
was monitored by the fluorescence increase with
ex = 550 nm and
em = 590 nm. The latter wavelengths minimized but
did not eliminate the contribution of F21 fluorescence to the HF31 fluorescence signal. The fluorescence changes were normalized to the
total fluorescence change for formation of enzyme-free F21 and
enzyme-free HF31. The time courses of the reaction with these
monitoring conditions were different (Fig. 8A). The increase in fluorescence from F21 (
ex = 492 nm) was a single
exponential process (k1 = 0.57 ± 0.01 s
1 and F1 =
0.2227 ± 0.0006). The
increase in fluorescence from F21 and HF31 (
ex = 550 nm)
was a double-exponential process (k1 = 0.57 s
1 (fixed), k2 = 0.18 ± 0.03 s
1, F1 = 0.10 ± 0.07, and
F2 =
0.43 ± 0.06). The early phase of this time
course was attributed to the fluorescence change associated with F21,
whereas the late phase was attributed to fluorescence changes
associated with HF31. These results suggested that dissociation of
E·HF31 was the last step of the catalytic cycle (Equation 10) (8).

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Fig. 8.
Dissociation of E·HF31 in the
presence of ATP. A, comparison of the time courses for
formation of free F21 and free HF31 from E·F21:HF31. ATP
(2000 µM) with 200 µg/ml poly(rU) was added to 50 nM E·F21:HF31. F21 was monitored with
ex = 492 nm and em = 550 nm, and HF31 was
monitored with ex = 550 nm and em = 590 nm. The solid line for the lower tracing was drawn with a
first-order rate constant of 0.57 s 1; the solid
line for the upper tracing was drawn with first-order rate
constants of 0.57 s 1 and 0.18 s 1.
B, effect of ATP on the dissociation of E·HF31.
The dissociation of E·HF31 was monitored with the
stopped-flow spectrofluorometer by the fluorescence increase upon
formation of HF31 ( ex = 492 nm and em
>530 nm). If the trap (5 µM 45-mer) and 700 µM ATP were added simultaneously to E·HF31
(generated by reaction of 50 nM E and 150 nM HF31 in the presence or absence of ATP), the time course
of the reaction was distinctly biphasic (inset, 0.64 s 1 and 0.19 s 1). The dependence of the
smaller rate constant on ATP concentration was determined from data
collected by simultaneous addition of ATP and trap to
E·HF31. Equation 3 was fitted to these data with
k 1 = 0.009 s 1, K = 130 µM, and k1 = 0.21 s 1 (solid line).
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To confirm that dissociation of E·HF31 was the late
phase in this reaction, the values of the rate constants for
dissociation of F21 and HF31 from the enzyme-DNA complex were
determined. The dissociation of E·HF31 and
E·F21 was monitored by the fluorescence changes
(
ex = 492 nm and
em >530 nm) associated
with formation of free HF31 and F21. Free enzyme was trapped with
excess (5 µM) 31-mer or 45-mer (TTT TTT ACA ACG TCG TGA
CTC TCT CTC TCT CTC TCT CTC TCT CTC). Both of these trapping agents
yielded similar results at several concentrations. In the absence of
ATP, the fluorescence change was a first-order process. The values of
the dissociation rate constants for E·F21 and
E·HF31 were 0.0345 ± 0.0004 s
1 and
0.009 ± 0.001 s
1, respectively, which were too
small for these processes to be competent in steady-state catalysis. If
the competitor DNA was added simultaneously with 2000 µM
ATP to E·F21 or E· HF31, the fluorescence
change was biphasic (Fig. 8B, inset). In the case of
E·HF31, addition of ATP to E·HF31 to form
"E·HF31·ATP" prior to initiating the reaction with
competitor DNA essentially eliminated the initial phase of the
reaction. In the absence of a DNA competitor to trap E as
E·HF31 dissociated, the amplitude of the late phase of the
reaction was very small (data not shown). Based on these observations,
the early phase of the fluorescence change was assigned to the reaction
of E·HF31 with ATP to form E·HF31·ATP as
described by Equation 11. The late phase of the fluorescence change was
assigned to the subsequent dissociation of
"E·HF31·ATP" to form E·ATP·DNA, where DNA was the competitor DNA. The kinetics of the early phase of
the reaction of ATP with E·HF31 were described by Equation 3 (k
1 = 0) with K = 150 ± 40 µM and k1 = 1.6 ± 0.2 s
1.
The first-order rate constants for dissociation of
E·HF31·ATP and E·F21·ATP (late phase of
the reaction) at a saturating concentration of ATP were 0.202 ± 0.003 s
1 and 1.52 ± 0.01 s
1,
respectively. The value of the rate constant for dissociation of
E·HF31·ATP was similar to the value of
kcat for unwinding F21:HF31 (0.12 s
1, Fig. 2), whereas the value of the rate constant for
dissociation of E·ATP·F21 was much too large to
contribute significantly to kcat.
Because the release of HF31 from E·HF31 could be a major
contributor to kcat for the unwinding of
F21:HF31, the effect of ATP concentration on the rate constant for
dissociation of E·HF31 (late phase of the reaction,
inset Fig. 8B) was investigated in more detail
(Fig. 8B). These data were described by Equation 3 with
k
1,ATP = 0.21 ± 0.01 s
1,
K = 90 ± 20 µM, and
k
1 = 0.009 s
1 (fixed), where
k
1,ATP (k1 in Equation 3) was the dissociation constant of E·HF31·ATP and
k
1 was the dissociation constant of
E·HF31.
F21:HF31 and 21:31 Interact with HCV Helicase
Similarly--
F21:HF31 was the double-stranded DNA used for most of
the experiments described herein. To ensure that our results were not significantly affected by the fluorescence labels on F21:HF31, selected
kinetic parameters were determined for the unlabeled duplex 21:31
(Table II). Comparison of the kinetic
parameters for the reaction of enzyme with F21:HF31 and 21:31 (Table
II) demonstrated that labeled and unlabeled double-stranded DNA were interacting similarly with the enzyme.
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Table II
Comparison of selected kinetic parameters for the reaction of HCV
helicase with F21:HF31, 21:31, and HF31
Kinetic parameters are defined in Equation #9.
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DISCUSSION |
Crystal structures for helicases from Bacillus
stearothermophilus (36), HCV (37), and E. coli (38) in
the absence of divalent metal ion indicated that they were monomeric in
the crystalline state. However, many helicases, such as E. coli Rep helicase (4), T4 helicase (32), and DnaB helicase (39),
appear to be oligomeric in the presence of substrates. In contrast to
these helicases, HCV helicase was a monomeric protein in the presence
of DNA and/or ATP analogues. Because HCV helicase contains a single
DNA-binding site per monomer (22), these results indicated that the
active form of this enzyme had a single DNA-binding site. Helicases
have been proposed to unwind duplex nucleic acids by either an
"active" or "passive" mechanism (7). The former mechanism
requires that the catalytically active form of the enzyme has multiple
nucleic acid-binding sites. Our finding that the active form of HCV
helicase was monomeric with a single nucleic acid-binding site
suggested that HCV catalyzed the unwinding of F21:HF31 by a passive
mechanism.
The goal of the present study was to identify the kinetic steps that
contributed to kcat. Unfortunately, relating
pre-steady-state data to the steady-state kcat
was complicated by the processivity of the enzyme, by the step size,
and by the number of base pairs broken for each ATP consumed. For
helicases, a simplified scheme describing the unwinding of duplex DNA
((bp)n) bound to the enzyme is given by Equation 12.
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(Eq. 12)
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In this simplified mechanism, the unwinding of double-stranded DNA
with n base pairs (bp) was considered to occur sequentially in n/m steps with each unwinding event described by the
pseudo first-order rate constant (k) that could be dependent
on ATP concentration. Furthermore, each partially unwound duplex could
dissociate from the enzyme and anneal in a process described by the
rate constant k'. This model is analogous to that described
by Ali and Lohman for E. coli helicase II (14). The
processivity of the enzyme for DNA (P) in this model is
given by Equation 13. The fraction of double-stranded DNA that was
separated into single-stranded DNA by HCV helicase in a single binding
event is given by Equation 14.
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(Eq. 13)
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(Eq. 14)
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An explicit expression for the time courses of strand separation
has been derived for the simple model of Equation 12 (14). In this
model, the reaction was initiated by addition of ATP and competitor DNA
to E·(bp)n. Competitor DNA ensured that partially
unwound (bp)n that dissociated from the enzyme annealed and did
not rebind to the enzyme. The time course for strand separation of
(bp)n (f(t)) is given by Equation 15.
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(Eq. 15)
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The assumptions for this derivation corresponded to experimental
conditions for the data of Fig. 5 where a competitor DNA was present to
trap free enzyme as F21:HF31 or products dissociated from the enzyme.
Numerical simulation of the time courses for strand separation of a
24-mer were made with step sizes equal to 1, 2, 3, 4, 6, 8, 12, and 24. A 24-mer was chosen over a 21-mer for these simulations because of the
greater number of possible step sizes evenly divisible into 24. The
simulated time courses for strand separation were biphasic in all cases
except for a step size of 24 base pairs. The initial phase of the
reaction using small step sizes was approximately linearly dependent on time, and the late phase of the reaction was exponentially dependent on
time. The lag time for the reaction (the time defined by the intersection of the line through the lag phase data and the line drawn
with the maximal slope through the exponential phase data (tl in Fig. 5)) was directly proportional to the
number of steps. More importantly, the time for transition from the
linear phase to the exponential phase (the portion of
tl that the time course had definite curvature,
tt in Fig. 5) was a decreasing fraction of the lag
time of the reaction. For example, the transition from the linear phase
to the exponential phase for the simulated data was 26, 46, and 62% of
the lag time for step sizes of 1, 2, and 3 base pairs, respectively.
The transition from the linear phase to the exponential phase for
unwinding F21:HF31 and 21:HF31 (Fig. 5 and Fig. 7) was very abrupt
(~25% of the total lag time). Based on the simple model above, these
results suggested that HCV helicase disrupted F21:HF31 at most several
base pairs at a time. Because the free energy of hydrolysis of ATP is
sufficient to break 2 to 4 base pairs (2, 7, 25), it was not
unreasonable to propose a step size of 2. This would result in the
efficiency for coupling the energy of ATP hydrolysis to base pair
breaking of over 50%. For a step size of 2 base pairs, a lag time of
approximately 1 s (average value from the data of Fig. 5 and 6),
and a duplex of 21 base pairs (F21:HF31), the value for the sum of
k and k' in Equation 12 was estimated from the
simulations described above to be approximately 7.5 s
1.
From the value for the sum of k and k' (7.5 s
1), the step size (m = 2), the fraction
unwound (0.44), and Equation 14, the values of k and
k' were estimated to be 7.0 s
1 and 0.5 s
1, respectively. The calculated value for dissociation
of F21:HF31 in various stages of unwinding from
E·ATP·F21:HF31 (k' = 0.5 s
1)
was in good agreement with the experimental measured value of 0.84 s
1 (Fig. 3). Furthermore, the values for P,
n/m, and k were used to predict the maximal value for
the effective rate constant (keff) for formation
of F21 from E·ATP·F21:HF31 in the presence of a saturating concentration of enzyme (Fig. 6). The expression for keff for conversion of (bp)n to
(bp)0 derived for the model of Equation 12 with
steady-state assumptions is given by Equation 16.
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(Eq. 16)
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Substituting estimated values for n/m (~11),
k (7 s
1), and P (0.93) into this
expression, the predicted value for keff was 0.43 s
1, which was similar to the observed value of 0.50 s
1 (Fig. 6).
Because the maximal value of the pseudo first-order rate constant for
formation of F21 from F21:HF31 with excess E and ATP (0.5 s
1) was 4-fold larger than the value of
kcat (0.12 s
1), another step must
contribute significantly to kcat. The
dissociation rate constant of E·ATP·HF31 was 0.21 s
1, which was similar to kcat. If
product dissociation and the unwinding step were the sole contributors
to kcat, the calculated value of
kcat (0.15 s
1) was very close to
the experimental value (0.12 s
1). Thus, the dissociation
of E·HF31 was the major contributor to
kcat for strand separation of F21:HF31 and the
unwinding steps were minor contributors to kcat.
The relevance of the interpretation of the kinetic results presented
herein for F21:HF31 to normal substrate was dependent on the assumption
that the tagged and untagged DNA substrates interacted similarly with
the enzyme. The similarity of the selected kinetic parameters for
interaction of the tagged and untagged DNA molecules with the enzyme
(Table II) suggested that this assumption was valid.
We gratefully acknowledge E. Furfine for
helpful discussions during the course of these studies.