From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101-6129
Received for publication, August 9, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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The mechanism of stimulation of a DNA
helicase by its cognate single-strand DNA-binding protein was examined
using herpes simplex virus type-1 UL9 DNA helicase and ICP8. UL9 and
ICP8 are two essential components of the viral replisome that associate into a complex to unwind the origins of replication. The helicase and
DNA-stimulated ATPase activities of UL9 are greatly elevated as a
consequence of this association. Given that ICP8 acts as a
single-strand DNA-binding protein, the simplest model that can account
for its stimulatory effect predicts that it tethers UL9 to the DNA
template, thereby increasing its processivity. In contrast to the
prediction, data presented here show that the stimulatory activity of
ICP8 does not depend on its single-strand DNA binding activity. Our
data support an alternative hypothesis in which ICP8 modulates the
activity of UL9. Accordingly, the data show that the ICP8-binding site
of UL9 constitutes an inhibitory region that maintains the helicase in
an inefficient ground state. ICP8 acts as a positive regulator by
neutralizing this region. ICP8 does not affect substrate binding, ATP
hydrolysis, or the efficiency of translocation/DNA unwinding. Rather,
we propose that ICP8 increases the efficiency with which substrate
binding and ATP hydrolysis are coupled to translocation/DNA unwinding.
We are using the UL9 DNA helicase of herpes simplex virus type-1
(HSV-1)1 and its cognate
single-strand DNA-binding protein (SSB), ICP8, to study the mechanism
by which SSBs stimulate DNA helicase-mediated DNA unwinding.
Stimulation of helicase activity due to specific association with an
SSB has been documented in numerous cases, but little is known about
the manner in which it occurs (for example see Refs. 1-8). It should
be noted, however, that, in some cases, stimulation of helicase
activity may not necessitate physical association of the SSB with the
helicase. UL9 and ICP8 are two HSV-1 gene products that are essential
for viral origin-dependent DNA replication (reviewed in
Refs. 9 and 10). UL9 is a sequence-specific DNA-binding protein of
~94 kDa that recognizes elements within the viral origins of
replication (11, 12). In addition, it possesses 3' to 5' helicase
activity and associated ATPase and single-stranded DNA (ssDNA) binding
activities (13-16). The minimum length of ssDNA required to stimulate
the ATPase activity of UL9 is 14 nucleotides (15). ICP8 is a ~128-kDa
protein that, relative to other known SSBs, binds ssDNA with low
affinity and low cooperativity (17). The site size of ssDNA binding is
10 ± 1 nucleotides (17).
ICP8 forms a specific and stoichiometric complex with UL9 by
interacting with its extreme C terminus (8, 18, 19). A complex of UL9
and ICP8 has been implicated in unwinding the viral origins of
replication (20, 21). In addition, ICP8 has been shown to greatly and
specifically increase the ssDNA-stimulated ATPase and helicase
activities of UL9, enhancing its processivity (8, 16). In contrast,
ICP8 has no effect on ATP hydrolysis in the absence of ssDNA cofactor,
indicating that it does not increase the chemical step of ATP
hydrolysis (8).
We previously hypothesized that ICP8 stimulates UL9 by tethering it to
the DNA template, thereby increasing its processivity (8). Here we show
that, in contrast to our hypothesis, the stimulatory effect of ICP8 is
not dependent on its ssDNA binding activity. To gain further insight
into the mechanism of stimulation, we examined the properties of a UL9
mutant that mimics ICP8-stimulated UL9. Moreover, we further
characterized the effects of ICP8 on the ATPase and helicase activities
of UL9 and two additional UL9 mutants that possess defects in DNA
unwinding. Our results indicate that ICP8 acts as a positive regulator
by neutralizing an inhibitory region of UL9 to allow more efficient
coupling between substrate binding and ATP hydrolysis and
translocation/DNA unwinding.
Materials--
-ATP (disodium salt), heparin-agarose,
phosphoenolpyruvate (potassium salt), and NADH were purchased from
Sigma. [
Bovine serum albumin (DNase-free) and T4 polynucleotide kinase were
obtained from Amersham Pharmacia Biotech and New England Biolabs,
respectively. Rabbit muscle L-lactic dehydrogenase and pyruvate kinase, as solutions in 50% glycerol, were obtained from Sigma. Proteinase K was purchased from Roche Molecular Biochemicals. Escherichia coli SSB and Sequenase version 2.0 DNA
polymerase were purchased from U.S. Biochemical Corp. UL9 (22), UL9DM27 (19), UL9C111A (22),
UL9C301A,2 and ICP8 (18) were
purified as described previously. Protein concentrations, expressed in
moles of monomeric protein, were determined using extinction
coefficients of 89,220 (UL9), 89,100 (UL9C111A and UL9C301A), 83,530 (UL9DM27), and 82,720 (ICP8) M
M13 mp18 virion DNA was purchased from New England Biolabs.
Oligodeoxyribonucleotides PB-11 (100-mer) (16), 60-mer (16), 20-mer
right (16), PB-9 (22-mer) (16), (dT)15 (8),
(dT)20 (8), (dT)60 (8), and 60-mer hairpin (8)
were as described. The 11-mer (PB-66, 5'-d(CCATGATTACG)) was obtained
from Operon Technologies. The helicase substrates consisting of
5'-32P-labeled 100-mer annealed to M13 ssDNA, and that
consisting of a 5'-32P-labeled 20-mer annealed to the 5'
side of a 60-mer, producing a partial DNA duplex with a 3' ssDNA
extension, were constructed as described previously (16). The helicase
substrate with strands of increasing length was produced by extending
M13 ssDNA primed with the UV Cross-linking--
Two nmol of ICP8 in 2 ml of 20 mM HEPES-NaOH, pH 7.6, 0.1 M NaCl, 10%
glycerol, 0.1 mM EDTA, and 1 mM dithiothreitol
was incubated at 4 °C for 10 min with 1 µM
(dT)15 prior to irradiation with 0.2 J/cm2 at
254 nm. Excess (dT)15 was removed from the reaction by
ultrafiltration through a 100,000 molecular weight cut-off membrane
(Eppendorf centrifugal filter tube). The concentrate was diluted to 1 ml with 20 mM HEPES-NaOH, pH 7.6, 0.1 M NaCl,
10% glycerol, 0.1 mM EDTA, and 1 mM
dithiothreitol and applied to a 500-µl heparin-agarose column
equilibrated with the same buffer. Under these conditions, ICP8 with
(dT)15 cross-linked to its ssDNA-binding site is not adsorbed while noncross-linked ICP8 is retained by the column. This
procedure was repeated three times to yield a preparation that is
enriched for ICP8 with (dT)15 cross-linked to its
ssDNA-binding site, henceforth referred to as cross-linked ICP8. The
protein was subsequently concentrated by ultrafiltration through a
30,000 molecular weight cut-off membrane (Eppendorf centrifugal filter tube) and the resulting preparation analyzed by SDS-polyacrylamide gel
electrophoresis followed by Coomassie Blue staining (Fig. 1). The ratio
of cross-linked to noncross-linked ICP8 in this preparation was
determined by densitometry using the Alpha Innotech AlphaImager 2000 imaging system.
Gel-mobility Shift Assay--
ICP8 was incubated with 1 nM (molecules) 5'-32P-labeled PB-9 in 10 µl
containing 25 mM EPPS-NaOH, pH 8.3, 2.5 mM
MgCl2, 1 mM dithiothreitol, 5% glycerol, and
100 µg/ml bovine serum albumin for 10 min on ice. The reactions were
mixed with 3 µl of 50% glycerol, 40 mM Tris acetate, pH
7.2, 1 mM EDTA, 0.25% bromphenol blue, and 0.25% xylene
cyanol, and resolved by electrophoresis through nondenaturing 4%
polyacrylamide-TBE gels at 100 V and 4 °C. Following electrophoresis, the gels were dried onto DE81 paper (Whatman). The
reaction products were analyzed and quantitated by storage phosphoranalysis with a Molecular Dynamics Storm 840. Binding affinities were determined by curve-fitting using the nonlinear cooperative ligand-binding equation of the GraFit version 4.09 program
from Erithacus Software.
Helicase Assay--
Helicase assays were performed essentially
as described (16). Unless otherwise stated, reactions were performed at
37 °C and contained 25 mM EPPS-NaOH, pH 8.3, 5.5 mM MgCl2, 3 mM ATP, 3 mM dithiothreitol, 10% glycerol, 100 µg/ml bovine serum
albumin, and DNA substrate and proteins as indicated. The reactions
were terminated by the addition of 0.3 volumes of 90 mM
EDTA, 6% SDS, 30% glycerol, 0.25% bromphenol blue, 0.25% xylene
cyanol, and 0.6 mg/ml proteinase K, followed by a 15-min incubation at
37 °C. The reaction mixtures were resolved by electrophoresis
through nondenaturing polyacrylamide-TBE gels. Following
electrophoresis, the gels were dried onto DE81 paper (Whatman).
Reactions were analyzed by autoradiography or quantitated by storage
phosphoranalysis with a Molecular Dynamics Storm 840.
ATPase Assay--
Rates of ATP hydrolysis were determined using
an enzyme-linked assay as described (8). Reactions were performed at
37 °C and contained 20 mM EPPS-NaOH, pH 8.3, 2.5 mM MgCl2, 2 mM
ATP/MgCl2, 200 µM NADH, 1.5 mM
phosphoenolpyruvate, 40 units/ml L-lactic dehydrogenase, 40 units/ml pyruvate kinase, 10 µM (nucleotide) DNA
cofactor, 3 mM dithiothreitol, 100 nM UL9 or
UL9DM27, and 500 nM ICP8 as indicated. Rates of ATP
hydrolysis were calculated by converting the absorbance change at 340 nm to moles of ATP hydrolyzed using an extinction coefficient of 6,220 M Modification of the ssDNA-binding Site of ICP8--
Since specific
residues required for the ssDNA binding activity of ICP8 have not been
identified, it is not possible to construct mutants that specifically
lack ssDNA binding activity. Consequently, ICP8 was covalently modified
by cross-linking with (dT)15. This method has previously
been documented and is specific for the ssDNA-binding site of ICP8
(25). (dT)15 slightly exceeds the length of ssDNA bound by
ICP8 (site size = 10 ± 1 nucleotides) (17) and should
therefore occupy its ssDNA-binding site and block subsequent ssDNA
binding. This is evident from the inability of cross-linked ICP8 to
bind heparin-agarose or ssDNA-cellulose (data not shown). Fig.
1 shows the composition of cross-linked ICP8 compared with UV-treated and untreated ICP8. Cross-linked ICP8 is
distinguished by its slightly slower electrophoretic mobility (25).
Densitometric analysis revealed that 33% of ICP8 in the preparation
was cross-linked to (dT)15. The ssDNA binding activity of
cross-linked ICP8 was measured directly using a gel-mobility shift
assay. Fig. 2 shows ssDNA-binding
isotherms for untreated ICP8, UV-treated ICP8, and cross-linked ICP8.
The amount of protein required for half-maximal ssDNA-binding was 0.1 µg for ICP8 and UV-treated ICP8 and 0.45 µg for cross-linked ICP8.
Consequently, cross-linked ICP8 exhibits 4.5-fold lower ssDNA binding
activity. It is interesting to note that cross-linked ICP8 showed lower ssDNA binding activity than would be expected from its degree of
cross-linking. This finding may be explained by the fact that some
unsubstituted ICP8 is also deficient in ssDNA binding activity and that
the preparation of cross-linked ICP8 is enriched for this form of ICP8
by repeated passage through heparin-agarose.
Requirement for the ssDNA Binding Activity of ICP8 in the
Stimulation of UL9--
To examine whether the stimulatory effect of
ICP8 depends on its ability to interact with ssDNA, cross-linked ICP8
was compared with untreated and UV-treated ICP8 in their ability to
stimulate the helicase activity of UL9. Fig.
3 shows that all three preparations of
ICP8 could stimulate UL9 to the same final extent (~9-fold stimulation) and that similar levels of stimulation were observed at
given protein concentrations. Given that the preparation of cross-linked ICP8 possesses 4.5-fold lower ssDNA binding activity, a
significant reduction in stimulatory activity should have been observed
if the ssDNA binding activity of ICP8 is indeed required for
stimulation. Fig. 3 also depicts two theoretical curves that show the
predicted stimulatory activity of ICP8 whose activity is reduced by
1.5-fold (based on the physical extent of cross-linking) or 4.5-fold
(based on the reduction in ssDNA binding activity).
To further examine the requirement for the ssDNA binding activity of
ICP8, the stimulatory effect of ICP8 on UL9 was examined in the
presence of a short (11-mer) oligodeoxyribonucleotide. In principle,
given that the ssDNA site size of ICP8 is 10 ± 1 nucleotides
(17), the 11-mer should occupy the ssDNA-binding site of ICP8 but is
too short to effectively interact with UL9 (15). Fig.
4 shows that addition of excess
concentrations of 11-mer (up to 50-fold more nucleotide than DNA
substrate and 110-fold higher than the concentration of UL9) had no
effect on the helicase activity of UL9, indicating that it is indeed
too short to compete with DNA substrate binding to UL9. More
importantly, excess concentrations of 11-mer (up to 50-fold more
nucleotide than DNA substrate, 50-fold higher than the concentration of
ICP8, and ~10-fold higher than the Kd for ssDNA
for ICP8 (Ref. 17)) did not reduce the stimulatory effect of ICP8. To
demonstrate that the 11-mer can indeed interact with ICP8 under these
conditions and therefore compete with DNA substrate binding, a variety
of gel-mobility shift assays were performed. Fig.
5A shows that the
concentrations of 11-mer that failed to disrupt the stimulatory effect
of ICP8 could in fact compete with 32P-labeled 22-mer in
the formation of a 22-mer-ICP8 complex. Quantitation of the data shows
that competition was ~99% effective at the highest concentration of
22-mer (Fig. 5B). Similarly, the 11-mer could compete with
M13 ssDNA in the formation of a M13 ssDNA-ICP8 complex and could itself
be bound by ICP8 in a gel mobility shift assay (data not shown).
Collectively, these data show that stimulation of DNA unwinding by ICP8
does not depend on its ability to interact with the DNA template.
Helicase Activity of UL9DM27--
UL9DM27 lacks the C-terminal 27 amino acids of UL9, which comprise the ICP8-binding site (19).
Consistent with the failure of UL9DM27 to associate with ICP8, its
helicase activity is not affected by ICP8 (19). Interestingly, UL9DM27
exhibits elevated ssDNA-stimulated ATPase and helicase activities
compared with wild-type UL9 (19). To establish whether the properties
of UL9DM27 mimic those of ICP8-stimulated UL9, we examined its
processivity using two different substrates. The first substrate,
consisting of strands of increasing length hybridized to M13 ssDNA, was
preincubated with UL9 or UL9DM27, and DNA unwinding was initiated by
the addition of ATP. At the same time, to capture helicase that
dissociated during the course of the reaction, a 5-fold molar excess of
M13 ssDNA was added. Furthermore, E. coli SSB was added to
trap unwound strands. Fig. 6A
shows a time course of DNA unwinding. The results indicate that,
although both UL9 and UL9DM27 catalyzed DNA unwinding to the
same final extent (products of >2.5 kilobase pairs), the rate
promoted by UL9DM27 was significantly faster.
The second substrate consisted of a 20-mer hybridized to the 5' end of
a 60-mer template strand. Because of the rapid kinetics of reannealing
of unwound 20-mer to the 60-mer template strand, optimal DNA unwinding
was only detected in the presence of at least a 5-fold molar excess of
unlabeled 20-mer to prevent reannealing of the labeled unwound strand
(data not shown). Substrate was preincubated with UL9 or UL9DM27, and
DNA unwinding was initiated by the addition of ATP. A time course of
DNA unwinding under single-turnover conditions ([enzyme] > [DNA])
shows that, although the final extent of DNA unwinding catalyzed by UL9
and UL9DM27 approached the same level, the rate promoted by UL9DM27 was
considerably faster (Fig. 6B). Moreover, addition of a
100-fold molar excess of a competitor 60-mer hairpin, which mimics the
structure of the DNA substrate, 1.5 min after the reaction was
initiated, inhibited further DNA unwinding by UL9. In contrast, there
was significant DNA unwinding after the addition of the competitor with
UL9DM27 (Fig. 6C). The faster rate of DNA unwinding by
UL9DM27 and its relative resistance to challenge with competitor
indicate that it possesses increased processivity as observed with
ICP8-stimulated UL9 (8).
ATPase Activities of UL9 and UL9DM27--
We have shown previously
that ICP8 has no effect on the ATPase activity of UL9 in the absence of
ssDNA cofactor (8). Similarly, the ssDNA-independent ATPase activity of
UL9DM27 is not elevated. In fact, the specific activity of ATP
hydrolysis in the absence of ssDNA cofactor (0.02 units/pmol, where 1 unit is that amount of enzyme that catalyzes the hydrolysis of 1 pmol
of ATP/s) is the same for both UL9 and UL9DM27. This finding further
supports the notion that UL9DM27 mimics the properties of
ICP8-stimulated UL9 and indicates that the increased activity
associated with deletion of the C-terminal 27 amino acids is not due to
enhanced ATP hydrolysis.
Rates of ATP hydrolysis for UL9 and UL9DM27 were determined in the
presence of (dT)20, (dT)60, and a 60-mer
hairpin, in the absence or presence of ICP8 (Fig.
7). Consistent with previous results for
UL9 (8), (dT)60 supported a higher rate of ATP hydrolysis
than (dT)20, indicating a correlation between translocation and ATP hydrolysis. The 60-mer hairpin possesses a 20-nucleotide 3'
ssDNA extension to serve as a loading site for UL9. In the absence of
DNA unwinding, it should support ATP hydrolysis to the same extent as
(dT)20. It is possible that, in the steady state, the rate
of ATP hydrolysis due to efficient utilization of the 60-mer hairpin
should be the sum of ATP hydrolysis required for DNA unwinding and that
due to translocation along unwound, linear 60-mer, and therefore exceed
that supported by (dT)60. For UL9, the rate of ATP
hydrolysis with the 60-mer hairpin was ~2-fold higher than with
(dT)20 but lower than with (dT)60. This intermediate level of ATPase activity is probably due to inefficient unwinding of the hairpin by UL9. The ATPase activity of UL9DM27 exhibited a similar dependence on ssDNA length, although its overall activity was greater than that of UL9. In contrast to UL9, UL9DM27 exhibited a higher rate of ATP hydrolysis with the 60-mer hairpin than
with (dT)60, indicating that it is more efficient at
utilizing this substrate. The addition of ICP8 stimulated the activity
of UL9 with (dT)20 and (dT)60. Interestingly,
ICP8 resulted in a higher rate of ATP hydrolysis with the 60-mer
hairpin than with (dT)60, indicating that it increases the
efficiency with which UL9 unwinds this substrate. The addition of ICP8
to reactions with UL9DM27 resulted in weak inhibition of ATP
hydrolysis, presumably because ICP8 can bind to the DNA and prevent
subsequent association of UL9DM27. As a conservative estimate, in the
steady state, the difference in ATP hydrolysis between
(dT)60 and the 60-mer hairpin reflects the amount of ATP
hydrolysis required for unwinding the hairpin. Interestingly, this
difference (1.3-fold) is identical for UL9DM27 and ICP8-stimulated UL9,
indicating equivalent efficiency.
The above studies on the helicase and ATPase activities of UL9DM27
indicate that it resembles ICP8-stimulated UL9. To examine whether the
stimulatory effect of ICP8 is due to changes in substrate (ATP and
ssDNA) affinity, the steady-state kinetic parameters kcat and Km were
determined for UL9, ICP8-stimulated UL9, and UL9DM27 (Table
I). The values for
kcat and Km for ATP and
ssDNA cofactor for UL9 are consistent with those reported previously (22, 26). As expected, ICP8-stimulated UL9 exhibits an increased kcat (~ 4-fold). However, there is no
significant change in the Km for ATP. Owing to
the fact that the ssDNA-stimulated ATPase activity of UL9 is inhibited
at high ICP8:ssDNA ratios, and the necessity to titrate
(dT)60 while keeping ICP8 concentrations constant, it was
not possible to determine Km for ssDNA cofactor for ICP8-stimulated UL9. Analysis of UL9DM27 shows that its
kcat is increased ~20-fold over UL9.
Interestingly, there are no significant changes in
Km for ATP or ssDNA cofactor for UL9DM27.
DNA-cross-linking was used to ascertain whether ICP8 had a significant
effect on the affinity for ssDNA. No significant increase in
cross-linking was observed with UL9 in the presence of ICP8 or with
UL9DM27 (data not shown).
Effect of ICP8 on UL9 Mutants with Defects in DNA
Unwinding--
UL9C111A and UL9C301A bear cysteine to alanine
substitutions in the vicinity of the "Walker" type A ATP-binding
motif, and helicase motif IV, respectively. UL9C111A shows a defect in
coupling ssDNA binding to high affinity ATP binding and subsequent
hydrolysis (22), while UL9C301A shows a defect in enzyme-DNA complex
formation.2 If ICP8 exerts its stimulatory effect at either
of these steps, these mutants should exhibit reduced stimulation in the
presence of ICP8. Fig. 8 shows that ICP8
stimulated UL9C111A and UL9C301A to the same level as wild-type
UL9.
The aim of this study was to further examine the mechanism by
which ICP8 stimulates the helicase activity of UL9. It was postulated previously that ICP8, due to its function as an SSB and its association with UL9, increases the processivity of UL9 by tethering it to the
DNA (8).
Two approaches were used to test the above prediction. First, ICP8 was
modified such that its ssDNA-binding site was occupied with an
oligodeoxyribonucleotide, preventing subsequent interactions with
ssDNA. This was achieved by UV cross-linking (dT)15 to ICP8 using a method that is specific for the ssDNA-binding site of ICP8
(25). Cross-linked ICP8 that was severely impaired in its ssDNA binding
activity was able to stimulate the helicase activity of UL9 to the same
levels as untreated or mock-treated ICP8. This finding clearly shows
that the stimulatory effect of ICP8 does not depend on its ability to
interact with the DNA template. In addition, the data also suggest that
cross-linked ICP8 is still competent to interact with UL9 since their
association is required for stimulation of DNA unwinding (19).
In the second approach, the ssDNA-binding activity of ICP8 was
modulated by selectively blocking its ssDNA-binding site with competitor DNA. This was achieved by incubating ICP8 with excess concentrations of 11-mer oligodeoxyribonucleotide. Consistent with the
results using cross-linked ICP8, excess 11-mer had no effect on the
ability of ICP8 to stimulate the helicase activity of UL9. Taken
together, these two approaches demonstrate that stimulation of UL9 does
not depend on the ability of ICP8 to interact with the DNA template. It
is conceivable that, to stimulate DNA unwinding, ICP8 must nevertheless
interact with ssDNA in trans (i.e. independent of
the DNA template). However, this would entail the unlikely scenario
that ssDNA activates ICP8, which in turn activates the helicase
activity of UL9. It is more feasible that the ssDNA- binding activity
of ICP8 is completely dispensable for its stimulatory effect, and that
ICP8 acts as an activator of UL9.
Previous studies have shown that maximal stimulation of helicase
activity occurs at a stoichiometry of 1:1 UL9:ICP8, consistent with the
existence of an equimolar complex (8). However, at this point it is not
known whether the active species consists of a dimer of UL9 and two
molecules of ICP8, or whether it involves an oligomeric species (16).
In any case, the results presented in this study show that the active
species does not require the ssDNA-binding activity of ICP8. However,
it is likely that, following DNA unwinding by the UL9-ICP8 complex,
stabilization of unwound DNA strands requires the ssDNA-binding
activity of ICP8. It is also possible that the overall stimulatory
effect of ICP8, especially that seen during the unwinding of long DNA
strands (16, 27), is a composite of the action of a UL9-ICP8 complex
(in which the ssDNA-binding activity of ICP8 is not required) and
excess ICP8 (to stabilize unwound DNA).
A complex of UL9 and ICP8 has been implicated in the unwinding of the
viral origins of replication (20, 21). Here ICP8 may serve as a
component of the active helicase and to stabilize unwound regions of
DNA. It is possible that the former function of ICP8 is independent of
its ssDNA-binding activity, as is the case for the unwinding of
nonspecific DNA, whereas the latter function would require its
ssDNA-binding activity. Consequently, it will be interesting to examine
whether ICP8 deficient in ssDNA-binding activity can substitute for
unmodified ICP8 during origin unwinding.
To further examine the mechanism by which ICP8 exerts its stimulatory
effect, we characterized the properties of a UL9 mutant (UL9DM27) that
lacks the C-terminal ICP8-binding site and therefore does not
physically interact with ICP8 or respond to its stimulatory activity
(19). Despite its inability to functionally or physically associate
with ICP8, it retains the properties of ICP8-stimulated UL9 (increased
ssDNA-stimulated ATPase and helicase activities, increased
processivity, and increased efficiency of DNA unwinding). These
observations suggest that the extreme C terminus of UL9 is an
inhibitory region that normally suppresses the enzymatic activities of
UL9. Furthermore, the role of ICP8 is to neutralize this inhibitory
region thereby activating UL9. Consequently, we conclude that ICP8
functions as a positive regulator that mediates its effect through the
C terminus of UL9.
To explain how ICP8 exerts its stimulatory effect, we entertained
several possibilities that were tested against our results. First, it
is possible that stimulation is brought about by improving the ratio of
ATP hydrolyzed per base pair unwound (i.e. increasing the
efficiency of DNA unwinding). Exact determination of the amount of ATP
required per base pair unwound is not practical due to limitations in
current technology. However, it is possible to calculate a ratio that
relates rates of ATP hydrolysis and DNA unwinding. We find that neither
ICP8-stimulated UL9 or UL9DM27 exhibit significant differences in this
ratio when compared with UL9. Consequently, it appears unlikely that
ICP8 acts in this capacity.
The second possibility involves a change in the step size of
translocation/DNA unwinding. It is possible that, without changing the
actual rate of movement along DNA, an increase in step size would lead
to an increase in the overall rate of translocation/DNA unwinding.
Given that the ssDNA-stimulated ATPase activity is a reflection of the
translocation of UL9 along ssDNA, an increase in step size would
actually decrease the effective length of DNA and result in a
concomitant decrease in the rate of ATP hydrolysis for a DNA cofactor
of a particular length. Moreover, since the stimulatory effect of ICP8
is substantial (~10-fold), a corresponding increase in step size
would have to be equally extensive and may not be within the limits of
a conformational change in UL9.
A third possibility is that ICP8 increases enzyme catalysis. This is
unlikely since neither ICP8-stimulated UL9 or UL9DM27 display elevated
ATPase activity in the absence of ssDNA cofactor.
The fourth possibility involves an increase in substrate affinity.
Accordingly, if substrate (ATP or ssDNA) is limiting, an increase in
affinity should enhance the rate of translocation/DNA unwinding. This
mechanism has been proposed for Schizosaccharomyces pombe
DNA helicase I, where RP-A increases the affinity of the enzyme for ATP
(6). Similarly, E. coli ribosomal protein L3 has been shown
to promote cooperative binding of Bacillus
stearothermophilus PcrA DNA helicase to its DNA substrate (7). It
is unlikely that ICP8 exerts its effect on UL9 in this manner since
neither ICP8-stimulated UL9 or UL9DM27 exhibit increased affinity for ATP or ssDNA.
Finally, the fifth possible mechanism by which ICP8 exerts its effect
is by increasing the efficiency with which substrate-binding (ATP and
ssDNA) and ATP hydrolysis are coupled to translocation/DNA unwinding.
In this case, ATP binding, ssDNA binding, and ssDNA-independent ATP
hydrolysis remain unaffected by ICP8, but more efficient coupling results in increased activity, which is reflected by increases in the
rates of ssDNA-stimulated ATP hydrolysis and DNA unwinding. This step
may entail a conformational change that allows UL9 to move along ssDNA
more easily. In light of the evidence against the earlier
possibilities, we favor this option. Precise determination of the
step(s) affected by ICP8 will require the use of rapid kinetic
techniques. Nevertheless, our present data with UL9C111A and UL9C301A,
which possess defects in coupling ssDNA binding to ATP binding and
subsequent hydrolysis, and ssDNA binding, respectively, suggest that
ICP8 exerts its effect downstream of these steps.
In conclusion, our data indicate that stimulation of UL9 is independent
of the ability of ICP8 to interact with the DNA template. Instead, ICP8
activates UL9 by binding to its extreme C terminus. Consequently, ICP8
acts as a positive regulator by neutralizing a region of UL9 that
otherwise suppresses the enzyme. Rather than increasing substrate
affinity, catalysis, efficiency of translocation/DNA unwinding, or step
size, ICP8 appears to increase the efficiency with which substrate
binding and ATP hydrolysis are coupled to translocation/DNA unwinding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (4,500 Ci/mmol) was from ICN.
1
cm
1 at 280 nm, or by the method of Bradford
(23) using bovine serum albumin as a standard.
40 sequencing primer in the presence of
dideoxynucleoside triphosphates and Sequenase version 2.0 DNA
polymerase as described previously (16).
1 cm
1
for NADH. Kinetic constants were determined using the nonlinear regression Michaelis-Menten kinetics curve-fitting program of Leatherbarrow (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Composition of cross-linked ICP8.
Coomassie Blue-stained 0.1% SDS-7.5% polyacrylamide gel of untreated
ICP8 (lane 1), UV-treated ICP8 (lane 2), and
cross-linked ICP8 (lane 3). The positions of ICP8 and
ICP8-(dT)15 are as indicated.
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Fig. 2.
ssDNA binding activity of cross-linked
ICP8. ssDNA binding activity was determined by a gel-mobility
shift assay as described under "Experimental Procedures."
A, untreated ICP8; B, UV-treated ICP8;
C, cross-linked ICP8.
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Fig. 3.
Stimulation of UL9 helicase activity by
cross-linked ICP8. Helicase activity was determined as described
under "Experimental Procedures," with 1 mM
dithiothreitol. UL9 (150 nM) was preincubated with 1 nM (molecules) M13:100-mer substrate for 20 min at
37 °C. The reactions were initiated by the addition of ATP and
untreated ICP8 (empty circle), UV-treated ICP8
(filled circle), or cross-linked ICP8
(filled square) and allowed to proceed for 15 min. The expected stimulatory activity of ICP8 that exhibits 1.5-fold
(short dashes) or 4.5-fold (long
dashes) reduced ssDNA binding activity is indicated. The
data represent the average of three independent experiments.
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Fig. 4.
Stimulation of UL9 helicase activity by ICP8
in the presence of ssDNA competitor. Helicase reactions were
performed essentially as described under "Experimental Procedures,"
with 20 mM EPPS-NaOH, pH 8.3, 1 mM
dithiothreitol, 5% glycerol, and 50 µg/ml bovine serum albumin. UL9
(150 nM) was preincubated with 0.5 nM
(molecules) M13:100-mer substrate for 20 min at 37 °C. The reactions
were initiated by the addition of ATP in the absence or presence of
326.5 nM ICP8 and 11-mer competitor as indicated, and
allowed to proceed for 15 min for UL9 (columns 1-4) or 5 min for UL9 and ICP8 (columns 5-8). Column 1,
UL9; column 2, UL9 and 326.5 nM (molecules)
11-mer; column 3, UL9 and 3.265 µM (molecules)
11-mer; column 4, UL9 and 16.325 µM
(molecules) 11-mer; column 5, UL9, ICP8 and 326.5 nM (molecules) 11-mer; column 6, UL9, ICP8 and
3.265 µM (molecules) 11-mer; column 7, UL9,
ICP8 and 16.325 µM (molecules) 11-mer; column
8, UL9 and ICP8.
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Fig. 5.
Interaction of 11-mer with ICP8. ssDNA
binding activity was determined by a gel-mobility shift assay as
described under "Experimental Procedures." A,
autoradiograph of reactions with 326.5 nM ICP8. Lane
1, no ICP8; lane 2, ICP8; lane 3, ICP8 and
326.5 nM (molecules) 11-mer; lane 4, ICP8 and
3.265 µM (molecules) 11-mer; lane 5, ICP8 and
16.325 µM (molecules) 11-mer. The positions of free
22-mer and of 22-mer-ICP8 complex are as indicated. B,
quantitation of the data shown in A. Column 1, ICP8;
column 2, ICP8 and 326.5 nM (molecules) 11-mer;
column 3, ICP8 and 3.265 µM (molecules)
11-mer; column 4, ICP8 and 16.325 µM
(molecules) 11-mer.
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Fig. 6.
Processivity of DNA unwinding. Helicase
activity was determined as described under "Experimental
Procedures." A, UL9 or UL9DM27 (175 nM) were
preincubated with 1 nM (molecules) substrate (M13 ssDNA
with strands of increasing length) for 10 min at 37 °C. The
reactions were initiated by the addition of ATP, 5 nM M13
ssDNA competitor, and 207 nM E. coli SSB
tetramer, and allowed to proceed for the times indicated.
Autoradiograph of a 4% polyacrylamide-TBE gel. Lane 1, DNA
substrate; lane 2, heat-denatured DNA substrate; lanes
3-7 and 8-12, reaction products after 5, 10, 20, 40, and 60 min with UL9 and UL9DM27, respectively. The positions of a
100-mer and 2.8-kilobase marker are as indicated. B, UL9
(empty circle) or UL9DM27 (empty
square) (100 nM) were preincubated with 10 nM (molecules) 60:20-mer substrate for 10 min at 37 °C.
The reactions were initiated by the addition of ATP and a 5-fold molar
excess of unlabeled 20-mer and allowed to proceed for the times
indicated. C, UL9 (filled circle) or
UL9DM27 (filled square) (100 nM) were
preincubated with 10 nM (molecules) 60:20-mer substrate for
10 min at 37 °C. The reactions were initiated by the addition of ATP
and a 5-fold molar excess of unlabeled 20-mer, and allowed to proceed
for the times indicated. After 1.5 min of incubation, 1 µM (molecules) 60-mer hairpin was added.
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Fig. 7.
ATPase activity of UL9 and UL9DM27: effects
of DNA length, structure, and ICP8. ATP hydrolysis was determined
as described under "Experimental Procedures." Reactions contained
10 µM (nucleotide) (dT)20 (columns
1, 4, 7, and 10),
(dT)60 (columns 2, 5, 8,
and 11), and 60-mer hairpin (columns 3,
6, 9, and 12). UL9 (columns
1-3 and 7-9); UL9DM27 (columns 4-6 and
10 - 12); ICP8 (columns 7-12).
Kinetic parameters of the ssDNA-stimulated ATPase activities of UL9,
ICP8-stimulated UL9, and UL9DM27
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Fig. 8.
Effect of ICP8 on DNA unwinding by UL9,
UL9C111A, and UL9C301A. Helicase activity was determined
essentially as described under "Experimental Procedures," with 1 mM dithiothreitol and 5% glycerol. One hundred and fifty
nM UL9 (columns 1 and 2), UL9C111A
(columns 3 and 4), or UL9C301A (columns
5 and 6) were preincubated with 1 nM
(molecules) M13:100-mer substrate for 20 min at 37 °C. The reactions
were initiated by the addition of ATP in the absence (columns
1, 3, and 5) or presence (columns
2, 4, and 6) of 82 nM ICP8 and
allowed to proceed for 15 min.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by Grant GM62643 from the National Institutes of Health and Grant 0050973B from the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Miami School of Medicine, P.O. Box
016129, Miami, FL 33101-6129. Tel.: 305-243-2934; Fax: 305-243-3955;
E-mail: pboehmer@molbio.med.miami.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M007219200
2 M. E. Arana and P. E. Boehmer, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HSV-1, herpes simplex virus type-1; EPPS, N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid); SSB, single-strand DNA-binding protein; ssDNA, single-stranded DNA.
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REFERENCES |
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