(Received for publication, September 24, 1996)
From the Department of Biochemistry, University of Arizona, Tucson, Arizona 85721-0088
The UL5, UL8, and UL52 genes of herpes simplex virus type 1 encode a multisubunit assembly that possesses primase, DNA helicase, and DNA-dependent nucleoside triphosphatase activities. A subassembly consisting of the UL5 and UL52 gene products retains these activities. The nucleoside triphosphatase activity of the UL5/UL52 subassembly is strongly stimulated by both homo- and heteropolymeric single-stranded DNA. Double-stranded DNA has little ability to stimulate the ATPase activity. The subassembly binds both double and single-stranded DNA. Nucleotides are not required for DNA-binding. The minimum length of single-stranded DNA that is bound and that stimulates enzymatic activity is about 12 nucleotides. The kinetic parameters of the ATPase activity of the subassembly are affected by the length of the oligonucleotide coeffector. The Km decreases as the coeffector length is increased up to a length of about 20 nucleotides and then remains independent of coeffector length. The first order rate constant for ATPase activity exhibits a quasihyperbolic dependence on the length of the DNA coeffector and is maximal for coeffectors of 20 nucleotides and longer.
Herpes simplex virus type 1 (HSV-1)1 encodes a heterotrimeric DNA helicase-primase whose subunits are encoded by the UL5, UL8, and UL52 genes of the virus (1, 2). The helicase activity of the enzyme is coupled to the hydrolysis of either ATP or GTP (1, 3). The UL5 gene product possesses a set of domains that correspond with several conserved motifs found in other DNA helicases (4). Mutagenesis of any one of these conserved domains in UL5 protein obliterates viral DNA synthesis (5). The UL52 gene is also essential for viral DNA synthesis and encodes a conserved domain that is found in other primases (6-8). Mutagenesis of this conserved domain specifically obliterates the primase activity of the HSV-1 helicase-primase (7, 8). Deletion of the UL8 gene from HSV-1 renders the virus incapable of DNA replication (9).
The HSV-1 helicase-primase can be isolated from insect cells that have been simultaneously infected with recombinant baculoviruses that express each of the three subunits that compose the holoenzyme (10). A subassembly consisting of the UL5 and UL52 gene products also exhibits the DNA-dependent NTPase, DNA helicase, and primase activities that are associated with the holoenzyme (11, 12). The primase activity of this subassembly exhibits DNA sequence dependence and is stimulated by the UL8 protein (13-15). However, purified UL8 protein itself lacks any type of discernible enzymatic activity (11, 12).
Homologs of the genes encoding the HSV-1 helicase-primase are found in several other human herpesviruses (16). The complex array of activities possessed by the helicase-primases encoded by this group of viruses may be attractive targets for antiviral drug design. An understanding of the properties of the HSV-1 helicase-primase may lead to strategies for developing such compounds.
In this work we further examine the interaction of the UL5/UL52 subassembly with various DNA coeffectors. Although the enzyme binds both double- and single-stranded DNA, double-stranded DNA fails to stimulate the NTPase activity of the enzyme to the same extent as does single-stranded DNA. Nucleotides are not required for DNA binding. Evidence is presented that shows that activation of NTPase activity varies with the length of the DNA coeffector and that the minimal DNA-binding site is about 12 nucleotides in length. The kinetic parameters for the ATPase activity of the subassembly are also affected by the length of the oligonucleotide coeffector and suggest that the full-length binding site is at least 20 nucleotides.
Deoxythymidine oligonucleotides were obtained
from U.S. Biochemical Corp. or Midland Certified Reagent Co. (Midland,
TX). The plasmid pVL941 (17) and M13mp18 single-stranded DNA were purified according to standard procedures (18). Nucleotides for the
NTPase assays were from Pharmacia Biotech Inc. Nucleotides for the
filter-binding assays were from Sigma. Bovine serum
albumin fraction V was from Sigma. Malachite green
hydrochloride and ammonium molybdate tetrahydrate, used in the
colorimetric DNA-dependent ATPase assay, were from
Sigma and Aldrich, respectively. Buffers were from
Sigma and U.S. Biochemical. DEAE and nitrocellulose membranes used for the filter binding assay were from Schleicher & Schuell. [-32P]ATP was from Amersham Corp. T4
polynucleotide kinase was from Life Technologies, Inc. Hydroxylapatite
(Bio-Gel HTP) was from Bio-Rad. The UL5/UL52 subassembly was purified
as described (12) except that hydroxylapatite fractionation was used in
place of the gel filtration step. Pooled fractions containing the
enzyme were loaded onto hydroxylapatite (1 mg of protein/ml of bed
volume) and eluted with a 10-400 mM sodium phosphate
buffer gradient, pH 7.5, containing 10% glycerol, 2 mM
dithiothreitol, 4 µg/ml pepstatin, and 4 µg/ml leupeptin. Fractions
containing the enzyme were pooled, diluted 4-fold in buffer containing
25 mM Tris-HCl, pH 8.0, 10% glycerol, 2 mM
dithiothreitol, 2 mM EDTA, 2 mM EGTA, 4 µg/ml
pepstatin, and 4 µg/ml leupeptin and applied to a 0.5-ml column of
DEAE. The column was then washed, and the enzyme was step-eluted with
equilibration buffer supplemented with 250 mM NaCl.
NTPase assays were essentially performed as described (1). Reaction mixtures (25 µl) contained 25 mM HEPES, pH 7.5, 3.5 mM MgCl2, 2.5 mM ATP, 10% glycerol, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, and the indicated amounts of DNA and the UL5/UL52 subassembly. For experiments in which the pH was varied, the following buffers were used in place of HEPES: PIPES, pH 6.5 and 7.0; EPPS, pH 8.0; TAPS, pH 8.5 and 9.0. Incubations were carried out at 37 °C for 30 min and then terminated by the addition of 0.75 ml of acidic molybdenum. Five minutes after termination, the absorbance at 650 nm was determined. The amount of inorganic phosphate that was released was determined by comparison with a standard curve in which 1 nmol of phosphate had an absorbance of 0.1.
Determination of Kinetic Parameters for ATPase ActivityThe NTPase assay described above was used with the following modifications. Reaction mixtures contained 32 nM UL5/U52, 30 µM oligonucleotide, and varying concentrations of ATP. The mixtures were incubated for 12 min at 34 °C and then terminated and developed as described above. The program DeltaGraph was used to calculate the Km and Vmax values from data that were directly fit to the Michaelis-Menten equation by nonlinear regression.
Filter Binding AssayNitrocellulose filter binding was
performed using the double filter method of Wong and Lohman (19). The
9.8-kilobase plasmid pVL941 was cleaved into 10 blunt-ended fragments
with HincII restriction enzyme. The fragments ranged in
length from 27 to about 4,600 base pairs. The fragments were
end-labeled using [-32P]ATP and T4 kinase using
standard procedures (18). Oligo(dT) ranging in length from 8 to 20 nucleotides was end-labeled similarly. Single-stranded DNA was
generated from the plasmid fragments by boiling the labeled DNA for 3 min followed by immersion in ice water. Binding reactions were set up
by adding the indicated quantities of the UL5/UL52 subassembly to
reaction mixtures (25 µl) containing 125 ng of end-labeled plasmid
DNA, 3.5 mM MgCl2, 30 mM NaCl, 10% glycerol, and 25 mM HEPES buffer, pH 7.5. Reactions that
used oligo(dT) contained 100 nM of the indicated
oligonucleotide and 67 nM of the UL5/UL52 subassembly.
Mixtures were assembled and incubated on ice for 1 h and then
filtered through the nitrocellulose-DEAE double membrane using a
Bio-Rad dot blot apparatus. The membranes had been conditioned as
described (19) and equilibrated in the binding buffer before use. The
filtered complexes were then washed with 200 µl of cold binding
buffer. Filtered complexes that were formed in the presence of
nucleotide were washed with buffer containing the same nucleotide at an
equivalent concentration. Binding of DNA to the DEAE and nitrocellulose
filters was quantitated simultaneously using a
-emission imaging
apparatus.
In order to examine the interaction of the UL5/UL52 subassembly with nucleic acids we first sought to identify pH and salt conditions that would yield maximal DNA-dependent ATPase activity. We also wanted to ascertain that conditions optimal for the ATPase activity of the subassembly are the same as those reported for the holoenzyme (20). We assumed that conditions that are optimal for the DNA-dependent ATPase activity of the enzyme would be optimal for interactions with nucleic acids.
The effect of pH on the DNA-dependent ATPase is shown in
Fig. 1a. The optimal pH was 7.5, and it
dropped abruptly on either side of the optimum. This result is similar
to that reported for the holoenzyme (20). Recent evidence suggests that
the HSV-1 helicase-primase has two distinct NTPase sites (3). One site hydrolyzes ATP only, and the other site hydrolyzes both GTP and ATP. We
therefore also examined the effect of pH on the GTPase activity of the
enzyme to determine if the two sites could be distinguished by
differences in their pH optima (Fig. 1a). The pH profile for
GTPase activity was the same as that for ATPase. Thus, if there are two
NTPase sites, their pH profiles are similar.
The effects of Mg2+ and various salts on the DNA-dependent ATPase activity of the UL5/UL52 subassembly were also examined. The optimal Mg2+ concentration for the DNA-dependent ATPase activity ranged from about 1 to 4 mM (Fig. 1b). These optimal Mg2+ concentrations are the same as those reported for the holoenzyme (20).
Different salts inhibited the DNA-dependent ATPase activity in a concentration-dependent manner (Fig. 1c). Eighty percent of maximal activity was observed in the presence of 50 mM acetate and chloride salts. At higher salt concentrations the chloride ion was more inhibitory than was acetate. The sulfate ion abolished activity at a concentration of 50 mM.
The Effect of Nucleic Acid Structure and Composition on the ATPase Activity of the UL5/UL52 SubassemblyThe
DNA-dependent NTPase activity of most DNA helicases is
stimulated by single-stranded DNA only. We sought to determine if this
is also true for the DNA-dependent ATPase activity of the
UL5/UL52 subassembly. We compared the effect of single-stranded DNA,
double-stranded DNA, and single-stranded RNA on the ATPase activity of
the enzyme. A 9.8-kb plasmid was cleaved into 10 blunt-ended fragments
that ranged from 27 to 4,600 base pairs in length. Mixtures containing
increasing amounts of the fragmented plasmid and a constant amount of
the UL5/UL52 subassembly were assayed for ATPase activity (Fig.
2a). In the presence of these double-stranded
DNA fragments, no activity was observed until the concentration of DNA
in the reactions exceeded 5 µM in nucleotide. The
fragments were next heat-denatured to yield single-stranded DNA and
then tested for the ability to stimulate the ATPase activity of the enzyme. Activity was observed at all concentrations of single-stranded DNA that were tested, with saturation occurring between 5 and 10 µM in nucleotide. At a single-stranded DNA concentration
of 5 µM, the ATPase activity was over 40-fold greater
than that observed in mixtures containing the double-stranded
fragments. A single-stranded homoribopolymer (poly(U)) failed to
stimulate the ATPase activity of the enzyme (Fig. 2b). Thus,
neither RNA nor double-stranded DNA serves as coeffector for the ATPase
activity of the UL5/UL52 subassembly.
We next compared the effects of homopolymeric and heteropolymeric single-stranded DNA on the ATPase activity of the UL5/UL52 subassembly. Heteropolymeric DNA is much less effective than homopolymeric DNA in stimulating the DNA-dependent ATPase activity of the HSV-1 origin-binding protein, which is also a DNA helicase and is encoded by the UL9 gene (21-23). Both poly(dT) and poly(dC) effectively activated the ATPase activity of the UL5/UL52 subassembly (Fig. 2b). However, poly(dA) was about 4-fold less effective as an activator. Poly(dT) and single-stranded M13mp18 DNA were equally effective in supporting the ATPase activity of the enzyme (Fig. 2c). This result contrasts with that observed for the HSV-1 origin-binding protein.
The Effect of Nucleotides on DNA BindingSome helicases, such
as the gene 4 protein encoded by bacteriophage T7, must first bind to a
specific nucleotide substrate before binding to DNA (24). Other
helicases, such as the Escherichia coli Rep helicase, do not
require a nucleotide cofactor for DNA binding (25). We used
nitrocellulose filter binding to assess the ability of the UL5/UL52
subassembly to bind to single-stranded and double-stranded plasmid DNA
fragments in the absence or in the presence of nucleotide. In the
absence of nucleotide, the subassembly bound both single- and
double-stranded DNA (Fig. 3a). However, the
enzyme bound single-stranded DNA about 5-fold more effectively than it
did double-stranded DNA. The effect of nucleotides on DNA binding was
examined next. To avoid complications of ATP hydrolysis on the binding
assay, the nonhydrolyzable ATP analog AMPP(NH)P was used to examine the
effect of ATP on DNA binding. Little effect on the binding of either
single-stranded or double-stranded DNA was observed in the presence of
2 mM AMPP(NH)P (Fig. 3b). The addition of 2 mM ADP to the mixtures increased the extent of binding to
single-stranded DNA but had little effect on binding to double-stranded
DNA (Fig. 3c). These results partially resemble those
observed for the effect of nucleotides on the binding of DNA by the
E. coli Rep helicase (26).
Activation of the NTPase Activity of the UL5/UL52 Subassembly Varies with the Length of the DNA Coeffector
Kinetic evidence
suggests that the HSV-1 helicase-primase has two distinct DNA-binding
sites that separately modulate the activity of the two putative NTPase
sites (3). We sought to determine if these two sites could be
distinguished based on differences in the size of the DNA binding
sites. We assumed that the size of the binding site(s) could be
estimated by determining the minimal length of single-stranded DNA that
activates the ATPase and GTPase of the UL5/UL52 subassembly.
Deoxythymidine oligonucleotides ranging in length from 6 to over 1000 residues were tested for the ability to activate the
DNA-dependent ATPase and GTPase activities of the enzyme
(Fig. 4, top). The activation profile was
similar for both ATPase and GTPase. Little activation of either ATPase
or GTPase was observed for oligonucleotides less than 12 residues in
length. Significant activation of both ATPase and GTPase was observed
when oligo(dT)12 was used as the coeffector. The extent of
activation continued to increase as the lengths of the oligo(dT) effector was increased and reached an apparent maximum for
oligonucleotides between 36 and 60 residues in length for both the
ATPase and GTPase activities.
We also used nitrocellulose filter binding to estimate the minimal length of DNA that is bound by the UL5/UL52 subassembly (Fig. 4, bottom). The minimum length that was bound was 10-12 nucleotides. The extent of binding to (dT)16 and (dT)20 was severalfold higher. Identical results were obtained by gel mobility shift analysis (data not shown). Thus, the minimal length of DNA that is effectively bound and that elicits NTPase activity is about 10-12 nucleotides. We conclude that, if there are two separate DNA binding sites that modulate the activity of the two putative NTPase sites, the sizes of these DNA binding sites are similar.
Effect of DNA Length on the Kinetic Parameters for ATPaseWe
next sought to determine under initial rate conditions whether the
progressive increase in ATPase activity that occurred with longer DNA
coeffectors is exerted through enhancement of the hydrolytic efficiency
or through the enhanced binding of the ATP substrate by the enzyme. The
ATPase activity of the UL5/UL52 subassembly was assayed in the presence
of constant concentrations of oligo(dT)n of increasing lengths
while the ATP concentration was varied. The Vmax
and Km were then determined and plotted as a
function of the length of the oligo(dT) activator (Fig.
5, a and b).
Vmax appeared to increase in a roughly linear manner as the length of the oligonucleotide was increased (Fig. 5a). However, the Km for ATP was constant
for oligo(dT) containing 20 or more residues (Fig. 5b). The
Km for ATP was higher in the presence of oligo(dT)
of lengths shorter than 20 nucleotides. The effect of the length of the
DNA coeffector on the first order rate constants
(Vmax/Km) showed a quasihyperbolic effect (Fig. 5c). The first order constant
appeared to reach a maximum for DNA coeffectors between 20 and 30 nucleotides in length and could be extrapolated to a minimum of about
10 nucleotides in length. These results suggest that the affinity for
ATP is progressively enhanced up to a critical length of the DNA
coeffector and then remains constant while the efficiency of catalysis
also increases and reaches a maximum once the DNA binding site is fully occupied.
In this work we have characterized the interaction of an active subassembly of the HSV-1 helicase-primase with various DNA coeffectors and have assessed their capacity to activate the nucleoside triphosphatase activity of the enzyme. We initiated this study by establishing conditions that maximize the NTPase activity of the UL5/UL52 subassembly. We assume that the pH, Mg2+, and salt optima for NTPase activity are also optimal for the binding of DNA coeffectors by the enzyme. The pH and Mg2+ conditions that we identified as optimal for the DNA-dependent NTPase activity of UL5/UL52 subassembly were the same as those reported for the holoenzyme (20). The range of pH that supports maximal DNA-dependent ATPase and GTPase is identical, with an optimum pH of 7.5. The pH optimum of the helicase-primase contrasts significantly with that of the UL9 protein, which is another HSV-1-encoded DNA helicase that is required for viral replication and has a pH optimum between 8.5 and 9.0 (23).
Kinetic evidence suggests that the HSV-1 helicase-primase has two sites that hydrolyze nucleoside triphosphates in a DNA-dependent manner. Both of these sites can apparently drive the unwinding of duplex DNA (3). The UL5 gene product contains a consensus nucleotide binding site near its amino terminus (27, 28). The location of the second putative NTP binding site on the enzyme is not known. It could reside either on the UL5 or the UL52 gene product. This second site would differ in structure from the consensus sequence at the UL5 N terminus and thus might have a different pH profile. Our failure to detect differences in the pH profiles for the DNA-dependent ATPase and GTPase activities of the enzyme suggests that there is either one DNA-dependent NTPase site, or, if there are two NTPase sites, their pH optima are similar.
The effect of nucleic acid composition and structure on the NTPase activity of the UL5/UL52 subassembly also differs from that of the UL9 protein. Double-stranded DNA functions poorly as a coeffector for the UL5/UL52 subassembly. However, double-stranded DNA containing specific sequences from the HSV-1 origin of replication are capable of eliciting the ATPase activity of the UL9 gene product.2 Single-stranded DNA is much more efficient than double-stranded DNA in stimulating the ATPase activity of the UL5/UL52 subassembly. Our observation that homopolymeric RNA is essentially inert as a coeffector suggests that recognition of the sugar-phosphate backbone is crucial for binding or for translocation of the enzyme along the DNA. Single-stranded homopolymers containing pyrimidines function just as well as heteropolymeric single-stranded DNA in stimulating the ATPase activity of the UL5/UL52 subassembly. In contrast to the UL9 protein, the HSV-1 helicase-primase utilizes heteropolymeric and homopolymeric single-stranded DNA equally well for activation of ATPase activity. Presumably the UL9 gene product, which functions as an origin-binding protein, is far less effective than the helicase-primase at unwinding regions of secondary structure in single-stranded heteropolymeric DNA. Furthermore, the DNA-dependent ATPase activity of the UL9 gene product is much weaker than that of the HSV-1 helicase-primase regardless of the type of single-stranded coeffector that is used.2 These distinctions may reflect different mechanisms of DNA unwinding by these two helicases, both of which have a different role in viral DNA replication.
The DNA binding properties of the UL5/UL52 subassembly differ from those of the helicase-primases encoded by the bacteriophages T4 and T7 (24). In contrast to the enzymes encoded by these phages, the binding of DNA by the herpes-encoded enzyme does not require prior binding of nucleotide. However, the affinity of the enzyme for DNA appears to be modulated by the presence of nucleotides in a manner that may be similar to that observed for the Rep helicase encoded by E. coli (26). Thus, the mechanism of DNA unwinding of the herpes enzyme may differ from the helicase-primases encoded by bacteriophages.
Recent evidence suggests that there are two separate DNA-binding sites on the HSV-1 helicase-primase that activate NTPase activity of the enzyme (3). One of these DNA-binding sites activates the NTPase site that hydrolyzes both GTP and ATP. The second DNA-binding site activates the site that hydrolyzes ATP only. We expected that the sizes of these two DNA-binding sites might differ and could be discriminated by differences in the lengths of poly(dT) that are required for NTPase activation. However, the minimal length of poly(dT) that stimulates the ATPase and GTPase activity of the UL5/UL52 subassembly is the same, about 12 nucleotides. The extent of stimulation for the ATPase and GTPase activities also appears to reach a maximum at about 36 nucleotides, which may reflect complete occupancy of the binding site. Tenney et al. (15) have obtained similar results for the stimulation of ATPase activity with heteropolymeric oligonucleotides. Thus, if there are two DNA-binding sites, the sizes of the binding pockets are similar.
The effect of the length of the DNA coeffector on the kinetic parameters for the ATPase activity of the UL5/UL52 subassembly is complex. The first order rate constant for the ATPase activity of the UL5/UL52 subassembly increases as the length of the DNA coeffector is increased and reaches a plateau for coeffectors of 20 nucleotides or longer, while the Km for ATP decreases and then remains constant for coeffectors of 20 nucleotides or longer. These observations could suggest that in the absence of DNA of a critical length the affinity of the helicase-primase for ATP is low. Upon binding DNA of a critical length, the enzyme shifts into a conformation that productively binds ATP. Further increases in the length of the DNA incrementally push the enzyme into a catalytically more efficient conformation until a maximum is reached at 20 nucleotides or longer. Young et al. (29) observed a somewhat similar phenomenon for the gene 41 helicase-primase encoded by bacteriophage T4. The extreme differences in the ability of poly(dT) versus poly(dA) to activate the ATPase activity of the helicase-primase suggests that steric variability in the nucleic acid sequence also affects the manner in which the DNA occupies the binding cleft. Consequently, the differences in catalytic efficiency with variable lengths and sequences of DNA may reflect a need for the binding site to be able to accommodate wide ranges of steric variability as the enzyme translocates along the DNA coeffector.
We thank Jennifer Hall, Nicoleta Constantin, and Savitha Devanathan for comments on the manuscript.