From the Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
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ABSTRACT |
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Herpes simplex virus type-1 UL9 protein is a sequence-specific DNA-binding protein that recognizes elements in the viral origins of DNA replication and possesses DNA helicase activity. It forms an essential complex with its cognate single-strand DNA-binding protein, ICP8. The DNA helicase activity of the UL9 protein is greatly stimulated as a consequence of this interaction. A complex of these two proteins is thought to be responsible for unwinding the viral origins of DNA replication. The aim of this study was to identify the mechanism by which ICP8 stimulates the translocation of the UL9 protein along DNA. The data show that the association of the UL9 protein with DNA substrate is slow and that its dissociation from the DNA substrate is fast, suggesting that it is nonprocessive. ICP8 caused maximal stimulation of DNA unwinding activity at equimolar UL9 protein concentrations, indicating that the active species is a complex that contains UL9 protein and ICP8 in 1:1 ratio. ICP8 prevented dissociation of UL9 protein from the DNA substrate, suggesting that it increases its processivity. ICP8 specifically stimulated the DNA-dependent ATPase activity of the UL9 protein with DNA cofactors that allow translocation of UL9 protein and those with secondary structure. These data suggest that UL9 protein and ICP8 form a specific complex that translocates along DNA. Within this complex, ICP8 tethers the UL9 protein to the DNA substrate, thereby preventing its dissociation, and participates directly in the assimilation and stabilization of the unwound DNA strand, thus facilitating translocation of the complex through regions of duplex DNA.
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INTRODUCTION |
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Herpes simplex virus type-1 (HSV-1)1 is a double-stranded DNA virus with a genome of ~152 kilobase pairs that contains three origins of DNA replication (1). Replication of origin-containing plasmids requires the action of seven viral gene products (2, 3). These seven gene products comprise a highly processive heterodimeric DNA polymerase (UL30/UL42 genes), a heterotrimeric DNA helicase-primase (UL5/UL8/UL52 genes), a single-strand DNA-binding protein (SSB) (UL29 gene), and an origin-binding protein (UL9 gene) (reviewed in Ref. 1).
The origin-binding protein (UL9 protein) is a 94-kDa protein that recognizes specific elements in the HSV-1 origins of DNA replication (4, 5). The UL9 protein also possesses intrinsic DNA helicase activity, and associated nucleoside triphosphatase (ATPase) activity, that is presumably required for unwinding the origins of DNA replication (6-9). The HSV-1 SSB, henceforth referred to as ICP8 (infected cell polypeptide 8), is a 128-kDa protein, capable of binding single-stranded DNA (ssDNA) cooperatively and with high affinity (10). ICP8 forms a specific complex with the UL9 protein by interacting with its extreme C terminus (11-13). In addition, ICP8 has been shown to interact with the HSV-1 DNA polymerase and helicase-primase (14-18). The ability of ICP8 to participate in multiple protein-protein interactions suggests that it fulfills several roles during viral DNA replication.
The interaction between ICP8 and UL9 protein greatly stimulates the rate and extent of DNA unwinding catalyzed by the UL9 protein, enabling it to unwind long stretches of DNA (9, 19). It has been shown that disruption of the ICP8-UL9 protein complex by deletion of the 27 C-terminal amino acid of the UL9 protein greatly reduces origin-specific DNA replication (12). Presumably, the ICP8-UL9 protein complex promotes efficient unwinding of the HSV-1 origins of DNA replication (20, 21).
This study examines the mechanism by which ICP8 stimulates the DNA helicase and ATPase activities of the UL9 protein. The results show that ICP8 increases the processivity of the UL9 protein, facilitating its translocation along DNA and through regions of secondary structure.
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EXPERIMENTAL PROCEDURES |
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Materials--
ATP (disodium salt), phosphoenolpyruvate
(potassium salt), and NADH (disodium salt) were purchased from Sigma.
[-32P]ATP (>5,000 Ci/mmol) was obtained from Amersham
Corp.
Proteins-- T4 polynucleotide kinase was obtained from Boehringer Mannheim and Promega. Bovine serum albumin (DNase-free) was purchased from Pharmacia Biotech Inc. Rabbit muscle L-lactic dehydrogenase and pyruvate kinase, as solutions in 50% glycerol, were obtained from Sigma. Escherichia coli SSB (E-SSB) was purchased from U. S. Biochemical Corp. E-SSB concentrations are expressed in moles of tetrameric protein.
ICP8 and UL9 protein were purified from Spodoptera frugiperda Sf21 cells infected with Autographa californica nuclear polyhedrosis virus recombinant for the HSV-1 UL29 and UL9 genes, respectively (11). The concentrations of ICP8 and UL9 protein were determined by using extinction coefficients of 82,720 and 89,220 MDNA Substrates--
M13 mp18 virion DNA and poly(dT) were
purchased from U. S. Biochemical Corp. Activated calf thymus DNA was
obtained from Sigma. (dT)15, (dT)20, and the
100-mer oligodeoxyribonucleotide (PB-11) (9) complimentary to residues
6208-6307 of the viral (+) strand of M13 mp18 DNA were obtained
from Operon Technologies. (dT)60 and the 60-mer hairpin
oligodeoxyribonucleotide
(5-d(GTCATGCTGACTAGTGTC-TTTTGACACTAGTCAGCATGAC (T)20)),
which possesses a 20-nucleotide 3
loading site for the UL9
protein, were obtained from the New Jersey Medical School Molecular
Resource Facility. The 38-mer and 53-mer oligodeoxyribonucleotides (22) were a gift from G. Villani (IPBS-CNRS, Toulouse, France). M13
mp18 ssDNA, activated calf thymus DNA, and poly(dT) concentrations were based on the manufacturers' specifications.
Oligodeoxyribonucleotide concentrations were determined using
extinction coefficients calculated from the DNA sequence using the
following formula: E 260 nm M
1
cm
1 = 0.89[(A × 15,480) + (C × 7,340) + (G × 11,760) + (T × 8,850)]. The 100-mer and 38-mer
oligodeoxyribonucleotides were 5
-32P-labeled using T4
polynucleotide kinase. DNA substrates for DNA helicase assays were
constructed by annealing 5
-32P 100-mer and 38-mer
oligodeoxyribonucleotides to M13 mp18 ssDNA and 53-mer
oligodeoxyribonucleotide, respectively (9, 22). Concentrations of the
DNA hybrids were based on the specific radioactivity of the labeled
oligodeoxyribonucleotides.
Enzyme Assays--
DNA helicase assays were performed
essentially as described (9). Unless otherwise stated, reactions were
performed at 37 °C and contained 25 mM EPPS-NaOH, pH
8.3, 2.5 mM MgCl2, 1 mM
dithiothreitol, 50 mM NaCl, 10% glycerol, 0.1 mg/ml bovine
serum albumin, and the indicated concentrations of UL9 protein, ICP8,
M13:100-mer, or 38:53-mer DNA substrates. Reactions containing the
38:53-mer DNA substrate also contained a 5-fold molar excess of
unlabeled 38-mer oligodeoxyribonucleotide to prevent reannealing of the unwound 5-32P 38-mer oligodeoxyribonucleotide. The
reactions were initiated by the addition of an equimolar solution of
ATP/MgCl2 to 3 mM and incubated for the times
indicated. The reactions were terminated by the addition of 0.3 volumes
of 90 mM EDTA, pH 8.0, 6% SDS, 30% glycerol, 0.25%
bromphenol blue, and 0.25% xylene cyanol. The reaction mixtures
containing M13:100-mer or 38:53-mer DNA substrates were resolved by
electrophoresis through 12% and 15% nondenaturing polyacrylamide-TBE
gels, respectively. Following electrophoresis, the gels were dried onto
DE81 paper (Whatman) and DNA unwinding quantitated by PhosphorImager
analysis.
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RESULTS |
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Effects of Preincubation and ICP8 on the DNA Helicase Activity of the UL9 Protein-- Consistent with previously published results (9), the time course of UL9 protein DNA unwinding shows an appreciable lag in the reaction (Fig. 1A). This lag period was eliminated upon preincubation of UL9 protein with DNA substrate (Fig. 1B). These results suggest that UL9 protein needs to assemble on the DNA substrate and that this association is slow and possibly rate-limiting.
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Effect of Competitor DNA on UL9 Protein DNA Unwinding in the Absence and Presence of ICP8-- Addition of a 3-fold molar excess of M13 mp18 ssDNA competitor to an ongoing DNA helicase reaction resulted in significant reduction of DNA unwinding activity (Fig. 3A). Furthermore, preincubation of the UL9 protein with DNA substrate to allow formation of a UL9 protein-DNA complex followed by addition of competitor DNA did not prevent competition (Fig. 3B). The ability of competitor DNA to reduce DNA unwinding activity, regardless of the prior formation of a UL9 protein-DNA complex, suggests that the UL9 protein DNA helicase is nonprocessive and that dissociation of the UL9 protein from the DNA substrate is fast.
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Effect of ICP8 on the ATPase Activity of the UL9 Protein-- Fig. 5 shows that ICP8 had no effect on the DNA-independent ATPase activity of the UL9 protein, suggesting that ICP8 does not directly affect the active site of the UL9 protein.
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Stimulation of the DNA-dependent ATPase Activity of the UL9 Protein by ICP8 Is Proportional to DNA Length and Is Affected by the Secondary Structure Content of the DNA Cofactor-- To examine the effect of ICP8 on the processivity of the UL9 protein, ATPase assays were performed with (dT) polymers of varying length (Fig. 7A). These DNA cofactors lack secondary structure and therefore allow unobstructed translocation of the UL9 protein along the DNA. Measurements on the effect of DNA length on the DNA-dependent ATPase activity of the UL9 protein showed that the minimum DNA length required to elicit activity is 14 nucleotides with activity increasing up to ~60 nucleotides (8). The data in Fig. 7A show that (dT)15 was sufficient to elicit the DNA-dependent ATPase activity of the UL9 protein. However, there was a significantly higher rate of ATP hydrolysis with (dT)20, (dT)60, and poly(dT) as cofactors, presumably due to the ability of the UL9 protein to bind and translocate along the longer DNA polymers while only capable of binding to (dT)15. Addition of ICP8 to a reaction with (dT)15 did not stimulate the rate of ATP hydrolysis. Similarly, ICP8 did not stimulate the rate of ATP hydrolysis with a 17-mer oligodeoxyribonucleotide (data not shown). In contrast, there was detectable stimulation of activity by ICP8 with (dT)20 and extensive stimulation with (dT)60 and poly(dT) (also see Fig. 8). The ability of ICP8 to stimulate ATP hydrolysis with DNA cofactors that allow translocation of UL9 protein suggests that ICP8 confers a higher degree of processivity upon the UL9 protein, presumably by preventing its dissociation from the DNA substrate.
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DISCUSSION |
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The aim of this study was to identify the mechanism by which the HSV-1 SSB, ICP8, stimulates the DNA helicase and DNA-dependent ATPase activities of the HSV-1 origin-binding protein (UL9 protein).
The results show that subsaturating concentrations of ICP8 were sufficient to stimulate the DNA helicase activity of the UL9 protein, and that maximal stimulation occurred at an ICP8 to UL9 protein ratio of 1:1. This observation is consistent with the existence of a specific ICP8-UL9 protein complex that translocates along the DNA and is active during DNA unwinding. In addition, the stoichiometry of 1:1 inferred from the functional interaction between ICP8 and UL9 protein is identical to that observed for the physical complex (13).
ICP8 also stimulated the DNA-dependent ATPase activity of the UL9 protein in a species-specific manner. A heterologous SSB, E-SSB, led to inhibition of activity, presumably by preventing access of the UL9 protein to the DNA substrate. Furthermore, ICP8 partially reversed the inhibitory effect of E-SSB. These observations are indicative of a specific tertiary complex that consists of UL9 protein, ICP8, and ssDNA.
Interestingly, the stimulatory effect of ICP8 on the DNA helicase and DNA-dependent ATPase activities of the UL9 protein was dependent on the ICP8/nucleotide ratio. Maximal stimulation was observed at subsaturating concentrations of ICP8, whereas coating concentrations produced less of an effect. Assuming that the level of stimulation correlates with the physical association of ICP8 and UL9 protein, these results suggest that the ICP8-UL9 protein complex is less stable at high ICP8/nucleotide ratios. This finding may explain why Gustafsson et al. (13) failed to detect a physical complex of UL9 protein, ICP8, and ssDNA at high ICP8/nucleotide ratios. It is possible that, when ICP8 is in excess and all ssDNA sites are occupied, ICP8 undergoes a conformational change and loses its affinity for UL9 protein.
The existence of a lag in the initial phase of the DNA unwinding reaction catalyzed by the UL9 protein suggests that the rate-limiting step is its association with the DNA substrate. This conclusion is substantiated by the observation that the lag was eliminated by preincubating the UL9 protein and DNA substrate. Moreover, rapid and maximal stimulation of DNA unwinding by ICP8 was only observed when UL9 protein was preincubated with DNA substrate or with ongoing DNA helicase reactions. Consequently, ICP8 exerts its maximal effect on a preassembled UL9 protein-DNA complex.
The observation that ICP8 had no effect on the DNA-independent ATPase activity of the UL9 protein suggests that it does not directly affect the catalytic site of the UL9 protein. Therefore, experiments were designed to examine how ICP8 influences the interaction of the UL9 protein with its DNA substrate during DNA unwinding and DNA-dependent ATP hydrolysis. Both activities entail translocation of the UL9 protein along ssDNA and through regions of duplex DNA. Consequently, these experiments addressed how ICP8 affects the translocation of UL9 protein along DNA.
Addition of excess challenger DNA to ongoing DNA unwinding reactions showed that UL9 protein was effectively competed from the DNA substrate, implying that it dissociates rapidly from the DNA substrate and that it is nonprocessive. The inhibitory effect of the competitor DNA was eliminated by ICP8. Specifically, equimolar, subsaturating concentrations of ICP8 added immediately prior to competitor DNA prevented the effect of the challenger DNA. These results suggest that ICP8 prevents the dissociation of UL9 protein from the DNA substrate and therefore increases its processivity. Further evidence for the processivity enhancing function of ICP8 is provided by previous experiments in which ICP8 enabled the UL9 protein to unwind long regions of DNA (up to ~3 kilobase pairs) (9, 19).
(dT)15 is sufficient to elicit the DNA-dependent ATPase activity of the UL9 protein (8). Presumably, this cofactor is sufficiently long to allow the UL9 protein to bind but is too short to allow translocation of the UL9 protein along the DNA. The extent of ATP hydrolysis seen with this cofactor should therefore be representative of UL9 protein binding only. Consistent with the assumption that ICP8 increases the processivity of the UL9 protein, there was no effect of ICP8 on the rate of ATP hydrolysis with (dT)15. In contrast, ATP hydrolysis with longer DNA cofactors, which allow both binding and translocation of the UL9 protein, was greatly stimulated by ICP8. Furthermore, ICP8 had an even greater stimulatory effect on ATP hydrolysis with DNA cofactors that contain secondary structure.
In conclusion, the ability of ICP8 to prevent competition with challenger DNA during DNA unwinding, and to stimulate ATP hydrolysis with DNA cofactors that allow translocation, implies that it prevents dissociation of the UL9 protein from the DNA substrate and increases its processivity. In addition, ICP8 appears to facilitate translocation of the UL9 protein through regions of duplex DNA. This is probably a manifestation of the helix-destabilizing activity of ICP8 (24).
Fig. 9 shows a model of how ICP8
stimulates the translocation of the UL9 protein along ssDNA and through
regions of duplex DNA. Both molecules within the UL9 protein dimer (6,
7, 19, 20) make contact with ssDNA via their N-terminal ssDNA-binding regions (25), enabling it to translocate 3 to 5
(7, 9). One molecule
of ICP8 is bound to the C terminus of each UL9 protein molecule (11,
12), allowing one ICP8 molecule to bind the template strand, tethering
the UL9 protein to the DNA substrate, while the second molecule of ICP8
assimilates and stabilizes the unwound primer strand, thereby
facilitating the translocation of the UL9 protein through regions of
duplex DNA.
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Previous studies have shown that a site-specific cisplatin lesion impairs the DNA helicase activity of the UL9 protein (22). Furthermore, it was shown that ICP8 relieved the inhibitory effect imposed by the lesion. Based on the findings in this study, it is likely that ICP8 enables the UL9 protein to bypass the lesion by tethering it to the DNA substrate, thereby preventing its dissociation.
The effects of cognate and noncognate SSBs have been documented for numerous DNA helicases (reviewed in Refs. 26-28). E-SSB has been shown to affect the activities of E. coli PriA (29, 30), DNA helicase IV (31), and Rep (32, 33) DNA helicases. Likewise, replication protein-A (RP-A) has been shown to affect several eukaryotic DNA helicases including: Saccharomyces cerevisiae Hel B (HCSB) (34, 35), calf thymus DNA helicases A-D and F (36-38), human DNA helicases isolated from HeLa cells (39, 40), and simian virus 40 large T antigen (41-43). In HSV-1, apart from the interaction between ICP8 and the UL9 protein discussed in this report, it has also been shown that ICP8 can specifically stimulate the DNA helicase activity of the DNA helicase-primase (17, 18, 44). In most cases, SSBs have been shown to enhance DNA unwinding by preventing nonproductive binding of the DNA helicase to the DNA substrate. Specific protein-protein interactions between DNA helicases and cognate SSBs have also been shown to increase the length of DNA unwound. However, no previous studies have addressed the exact mechanism by which an SSB stimulates DNA unwinding.
Previous studies have indicated the importance of the ICP8-UL9 protein interaction for HSV-1 origin-specific DNA replication (12, 21). The properties of the UL9 protein and ICP8 and those of the ICP8-UL9 protein complex, described in this and previous studies (7, 9, 10, 20, 21, 24), illustrate how this complex is suited for its predicted role of recognizing and unwinding the HSV-1 origins of DNA replication.
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FOOTNOTES |
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* This work was supported by Grant AI 38335 from the National Institutes of Health.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.
1
The abbreviations used are: HSV-1, herpes
simplex virus type-1; SSB, single-strand DNA-binding protein; ssDNA,
single-stranded DNA; E-SSB, E. coli SSB; EPPS,
N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid).
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REFERENCES |
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