Cysteine 111 Affects Coupling of Single-stranded DNA Binding to ATP Hydrolysis in the Herpes Simplex Virus Type-1 Origin-binding Protein*

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.

The UL9 protein also catalyzes the hydrolysis of ATP, which is greatly stimulated by single-stranded DNA (ssDNA), reflect-ing the ability of the protein to translocate along ssDNA and to unwind DNA with a polarity of 3Ј to 5Ј (18 -22). The ATPase and DNA helicase activities as well as a distinct ssDNA-binding activity localize to the N-terminal two-thirds of the UL9 protein (residues 1-534) (23). Mutations in the ATP-binding site or the conserved DNA helicase motifs of the UL9 protein abolish origin-dependent replication in a transient system (24,25).
The function of the UL9 protein is to initiate replication by unwinding the DNA at the origins (26,27). This function is probably performed in conjunction with the HSV-1 single strand DNA-binding protein (ICP8) which interacts with the C-terminal domain of the UL9 protein (28). This interaction is important for origin-dependent replication and has been shown to stimulate the DNA helicase activity of the UL9 protein by preventing its dissociation from the DNA substrate (29,30). Presumably, the sequence-specific DNA-binding activity of the UL9 protein targets a complex of UL9 protein and ICP8 to the origins to promote efficient DNA unwinding (1,27).
The UL9 protein contains a remarkably high content of cysteines (24 out of 851 amino acids) that are randomly distributed throughout the primary sequence (7). None of these cysteines are arranged into motifs that are involved in metal binding such as zinc fingers or RING fingers. Thus far there have been no reports on the importance of cysteines for the activities of the UL9 protein. In this study, we show that the UL9 protein contains N-ethylmaleimide (NEM)-sensitive cysteines that overlap with the ssDNA-binding site and are involved in signal transduction between ssDNA-binding and ATP hydrolysis and movement of the UL9 protein along DNA. The inability to identify a specific cysteine that is modified by NEM prompted us to use site-directed mutagenesis to identify the cysteine that is responsible for the inhibitory effect of NEM. Since the ATPase and DNA helicase activities of the UL9 protein reside in the N-terminal domain (23), the susceptible cysteine(s) must be in this region. Although this part of the UL9 protein contains 17 cysteines, only four of these are perfectly conserved in the origin-binding proteins of at least 10 different herpesviruses. Since amino acids that perform critical tasks are likely to be conserved, these cysteines may be responsible for NEM-induced inactivation of the UL9 protein. One of these cysteines (cysteine 111) is located in the vicinity of the "Walker" type A adenine nucleotide-binding site and DNA helicase motif I ( Fig. 1) (31)(32)(33) and may therefore represent the cysteine responsible for NEM-induced inactivation of the UL9 protein.
Our results indicate that cysteine 111 is an essential residue that is involved in coupling ssDNA-binding to ATP hydrolysis, which represents a key step in the mechanism of action of a DNA helicase.
Mutagenesis of UL9 Cysteine 111-Site-directed mutagenesis of cysteine 111 to alanine was performed with a modification of the Quick-Change procedure from Stratagene. The template for the mutagenesis reaction was pSL1180UL9, which contains the 3.4-kilobase pair NdeI-SspI UL9-encoding fragment from pET-3a/OBP (10) inserted into the NdeI and StuI sites of pSL1180 (Amersham Pharmacia Biotech). The mutagenesis reaction contained 25 ng of pSL1180UL9, 0.5 M (molecules) mutagenic primers PB-83 (5Ј-CGTCGTCTCCGCTCGTCGGAGT) and PB-84 (5Ј-ACTCCGACGAGCGGAGACGACG), 5% dimethyl sulfoxide, and 2.5 units of PfuTurbo DNA polymerase. Following denaturation of the template at 95°C for 4 min, the primers were extended for 16 cycles (95°C for 30 s, 60°C for 1 min, and 72°C for 5 min). After an additional 10 min at 72°C, the reaction mixture was treated with 10 units of DpnI for 105 min to digest the parental DNA. The digested DNA was used to transform Escherichia coli JM109 cells. Progeny plasmids were screened for the presence of the mutation by DNA sequencing. Progeny plasmid with the desired mutation was designated pSL1180UL9C111A.
Expression of UL9C111A Protein in Spodoptera frugiperda Cells-S. frugiperda Sf9 and Sf21 cells were maintained at 27°C in Sf900 II-serum free medium (Life Technologies, Inc.). Recombinant Autographa californica nuclear polyhedrosis virus (NPV) expressing the UL9C111A gene was generated using the Bac-To-Bac baculovirus expression system from Life Technologies, Inc. Briefly, pSL1180UL9C111A was digested with NotI and XbaI. The 3.4-kilobase pair UL9C111A-encoding fragment was inserted into the NotI and XbaI sites of pFastBac1 (Life Technologies, Inc.) to generate pFastBacUL9C111A. pFastBacUL9C111A was transformed into E. coli DH10Bac cells (Life Technologies, Inc.), and colonies harboring recombinant bacmids were selected. Purified bacmid DNA was transfected into Sf9 cells. Seventy-two hours post-transfection, the cell culture media containing recombinant A. californica NPV were collected. Following a round of amplification, recombinant A. californica NPV isolates were used to screen for expression of UL9C111A protein in Sf9 cells infected at a multiplicity of infection (m.o.i.) of 10. A clone of A. californica NPV that expressed high levels of UL9C111A protein was selected for further studies.
Proteins-Bovine serum albumin (DNase-free) and PfuTurbo DNA polymerase were obtained from Amersham Pharmacia Biotech and Stratagene, respectively. The Klenow fragment of E. coli DNA polymerase I and T4 polynucleotide kinase was purchased from Roche Molecular Biochemicals and Promega, respectively. Rabbit muscle L-lactic dehydrogenase and pyruvate kinase, as solutions in 50% glycerol, were obtained from Sigma. UL9C111A protein was purified from Sf21 cells infected with A. californica NPV recombinant for the UL9C111A gene. One liter of Sf21 cells at 2 ϫ 10 6 cells/ml in Sf900 II serum-free medium supplemented with 1% fetal calf serum was infected at a m.o.i. of 5.
Sixty hours postinfection, the cells were harvested. UL9C111A protein was purified from nuclear extracts essentially as described for UL9 protein (28) with the following modifications. UL9C111A protein, eluting from heparin agarose at ϳ650 mM NaCl, was dialyzed twice against 2 liters of 10 mM sodium phosphate pH 7.2, 0.1 M NaCl, 10% glycerol, and 1 mM dithiothreitol (DTT) prior to chromatography on hydroxyapatite. Hydroxyapatite chromatography was performed as described for UL9 protein (28), except that the gradient was from 10 to 200 mM sodium phosphate, pH 7.2. UL9C111A protein, eluting at ϳ100 mM sodium phosphate, was subsequently purified on a 1-ml Resource S column (Amersham Pharmacia Biotech) as described for UL9 protein on a MonoS HR 5/5 column (28), except that DTT was omitted from the buffer. The peak of UL9C111A protein, containing nearly homogenous (Ͼ95% pure) protein, eluted at the same position as UL9 protein, corresponding to ϳ370 mM NaCl. Fig. 2A shows the elution of UL9C111A protein from the Resource S column. The yield of purified UL9C111A protein was ϳ0.2 mg. UL9 protein was purified from Sf21 cells infected with A. californica NPV recombinant for the UL9 gene as described for UL9C111A protein above. Protein concentrations, expressed in mol of monomeric protein, were determined using extinction coefficients of 89,220 and 89,100 M Ϫ1 cm Ϫ1 at 280 nm for UL9 and UL9C111A proteins, respectively.
DNA-M13 mp18 virion DNA was purchased from U.S. Biochemical Corp. The 22-mer oligodeoxyribonucleotide PB-9 and (dT) 60 were as described (20,30). The DNA helicase substrate was constructed by annealing 5Ј-32 P-labeled PB-9 to M13 mp18 ssDNA (20). Its concentration was based on the specific radioactivity of the 5Ј-32 P-labeled PB-9. The DNA substrate for the origin-binding assay was a 176-base pair EcoRI fragment that contains a copy of wild-type HSV-1 oriS and was 3Ј-32 P-labeled with the Klenow fragment of E. coli DNA polymerase I and [␣-32 P]dATP. The oriS-containing plasmid pGEM822 was constructed by inserting the 822-base pair BamHI oriS fragment from pOS-822 (34) into the BamHI site of pGEMEX (Promega).
NEM Modification of UL9 and UL9C111A Proteins-Unless otherwise stated, UL9 or UL9C111A proteins (8 -23 pmol) were modified with the indicated concentrations of NEM in 20 mM HEPES-NaOH, pH 7.0, for 10 min at room temperature. In reactions containing ssDNA, UL9 protein was preincubated with 16 M (nucleotide) (dT) 60 for 5 min on ice prior to modification with NEM. The reactions were quenched by the addition of DTT to 5 mM followed by 5 min at room temperature. Similar reactions lacking UL9 protein were performed in parallel to neutralize the NEM. The activities of UL9 protein were examined with neutralized NEM to control for any inhibitory impurities in the preparation of NEM. NEM modification had no effect on the electrophoretic mobility of the UL9 protein in SDS-polyacrylamide gels (data not shown). To determine the stoichiometry of NEM modification, 1 nmol of UL9 protein was incubated with 1 mM N-[ethyl-1,2-3 H]maleimide (74.2 mCi/mmol) in 20 mM HEPES-NaOH, pH 7.0, and 0.1 M NaCl for 10 min at room temperature. The reaction was quenched by the addition of DTT to 10 mM followed by a 5-min incubation at room temperature. NEM-modified UL9 protein was dialyzed three times against 2 liters of 0.1 M (NH 4 )HCO 3 . The stoichiometry of NEM modification was calculated based on the specific radioactivity of NEM and the radioactivity associated with the UL9 protein following dialysis.
ATPase Assay-ATP hydrolysis was measured with two different assays. The effect of DTT on the DNA-dependent ATPase activity of the UL9 protein was measured in reactions (25 l) containing increasing concentrations of DTT (0 -10 mM) and 20 mM EPPS-NaOH, pH 8.3, 50 mM NaCl, 4.5 mM MgCl 2 , 2 mM ATP, 10 M (nucleotide) (dT) 60 , and 0.1 mg/ml bovine serum albumin in the absence or presence of 100 nM UL9 protein. Reactions were incubated for 30 min at 37°C followed by the addition of 750 l of acidic ammonium molybdate solution containing malachite green to measure the formation of inorganic phosphate (35). After 5 min of color development at room temperature, the absorbance at 650 nm was determined. Rates of ATP hydrolysis were determined using an enzyme-linked assay as described (30). Unless otherwise stated, reactions were performed at 37°C and contained 20 mM EPPS-NaOH, pH 8.3, 4.5 mM MgCl 2 , 2 mM ATP, 200 M NADH, 1.5 mM phosphoenolpyruvate, 40 units/ml L-lactic dehydrogenase, 40 units/ml pyruvate kinase, 10 M (nucleotide) (dT) 60 , 100 nM UL9 or UL9C111A proteins, and DTT as indicated. Rates of ATP hydrolysis were calculated by converting the absorbance change at 340 nm to mol of ATP hydrolyzed using an extinction coefficient of 6,220 M Ϫ1 cm Ϫ1 for NADH at 340 nm. Kinetic constants were determined using the nonlinear regression Michaelis-Menten kinetics curve-fitting program of Leatherbarrow (36).
ssDNA Binding Assay-The ssDNA-binding activity of the UL9 protein was measured by a gel mobility shift assay essentially as described (23). Five picomoles of UL9 protein, UL9 protein with neutralized NEM, or NEM-modified UL9 protein were incubated with 60 fmol of 5Ј-32 Plabeled PB-9 DNA in 21 l containing 20 mM HEPES-NaOH, pH 7.0, 0.1 M NaCl, 2.5 mM MgCl 2 , and 5 mM DTT for 10 min on ice. The reactions were mixed with 4 l of 80% glycerol containing 0.25% bromphenol blue and resolved by electrophoresis through nondenaturing 12% polyacrylamide-TBE gels at 100 V and 4°C. Following electrophoresis, the gels were dried onto DE81 paper (Whatman), and the fraction of bound DNA was quantitated by PhosphorImager analysis.
Origin Binding Assay-Origin binding activity was measured by nitrocellulose filter binding essentially as described (8). UL9 and UL9C111A proteins were incubated with 15 fmol of 32 P-labeled oriS DNA in 25 l containing 10 mM HEPES-NaOH, pH 7.5, 5 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 100 g/ml bovine serum albumin, and 25 g/ml poly(dI-dC)⅐poly(dI-dC) for 5 min at room temperature followed by 10 min on ice. The reactions were diluted with 1 ml of ice-cold 25 mM HEPES-NaOH, pH 7.5, 0.1 M NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, and 10% glycerol and immediately filtered through 0.45-m Millipore type HA filters that had been equilibrated in the same buffer. Filtration was complete within 10 s. The filters were dried and counted in 4 ml of ScintiSafe 30% liquid scintillant (Fisher). The results were corrected for background binding observed in the absence of origin-binding protein and quantitated based on the specific radioactivity of the 32 P-labeled oriS DNA.
oriS-dependent DNA Replication in Vivo-oriS-dependent DNA replication in Sf9 cells was determined essentially as described (5,29). 3 ϫ 10 5 Sf9 cells were seeded in 24-well Linbro plates (2 cm 2 ) and transfected with 150 ng of pGEM822 using a Cellfectin (Life Technologies, Inc.)-mediated protocol. The cells were allowed to recover for 24 h and either mock-infected or infected with recombinant A. californica NPV that encoded the HSV-1 UL5 (37) Sixty-five hours postinfection, the cells were harvested, and total cellular DNA was extracted using the DNeasy Tissue kit from Qiagen. 4.5 g of DNA was digested with 7.5 units of HindIII for 16 h to linearize pGEM822. Half of the sample was further digested with 7.5 units of DpnI for 16 h to digest unreplicated DNA. The DNA samples were resolved by electrophoresis through 1% agarose-TAE gels followed by transfer of the DNA to a Trans-blot 0.2-m nitrocellulose membrane (Bio-Rad). pGEM822 DNA was detected by Southern blot hybridization using random-primed (Life Technologies, Inc.) 32 P-labeled pGEM822 as a probe. Experiments were performed in quadruplicate.

Effects of Dithiothreitol and N-Ethylmaleimide on the UL9
Protein-DTT stimulated the DNA helicase and DNA-dependent ATPase activities of UL9 protein up to a maximum of 5-and 2-fold, respectively (Table I). Incubation of UL9 protein with 1 mM NEM rapidly (within 30 s) inactivated its DNA helicase activity (Fig. 3). Similarly, NEM inactivated the DNA-depend-  ent ATPase activity of UL9 protein but had no significant effect on its DNA-independent ATPase, ssDNA-binding, and origin binding activities ( Table I) Incubation of UL9 protein with ssDNA ((dT) 60 ) fully protected its DNA-dependent ATPase activity from NEM inactivation (Fig. 4). In addition, both ATP (5 mM) and duplex DNA (15 M nucleotide) also provided partial protection from NEM inactivation (data not shown). Using N-[ethyl-1,2-3 H]maleimide, the stoichiometry of modification was determined as 14:1 NEM: UL9 protein, indicating that the majority (14 out of 24) of cysteines in the UL9 protein are susceptible to modification by NEM. In the presence of (dT) 60 , this ratio was decreased to 2:1 NEM:UL9 protein, indicating that the majority (12 out of 14) of the NEM-susceptible cysteines are protected by ssDNA.
Purification of UL9C111A Protein-A UL9 protein variant, designated UL9C111A protein, in which cysteine 111 is substituted with alanine, was expressed and purified to near homogeneity from A. californica NPV-infected Sf21 cells. The purity of the final protein preparation, eluting from a Resource S column, is shown in Fig. 2A. The identity of purified UL9C111A protein was confirmed by immunoblot analysis with an anti-UL9 protein rabbit serum (Fig. 2B).
Characterization of the ATPase and DNA Helicase Activities of UL9C111A Protein-The rate of ATP hydrolysis by UL9C111A protein was compared with that of UL9 protein both in the absence and presence of a ssDNA cofactor, (dT) 60 . Fig. 5A shows that the rate of DNA-independent ATP hydrolysis by UL9C111A protein did not significantly differ from that of UL9 protein. In contrast, UL9C111A protein exhibited Ͻ50% of the rate of DNA-dependent ATP hydrolysis observed with UL9 protein (Fig. 5B). The rate of DNA unwinding exhibited by UL9C111A protein was also reduced to ϳ50% of that of UL9 protein (Fig. 6A). Likewise, the specific activity of DNA unwinding of UL9C111A protein was approximately half that of UL9 protein (Fig. 6B).
Mutagenesis of Cysteine 111 to Alanine Does Not Affect the Stability of the UL9 Protein or Its Origin Binding Activity-To confirm that UL9C111A protein is not structurally altered, we compared its thermal stability to that of UL9 protein. The thermal inactivation curves of UL9 and UL9C111A proteins, both in the absence and presence of (dT) 60 , were the same (data not shown). Similarly, substitution of cysteine 111 with alanine had no effect on the origin binding activity of the UL9 protein (data not shown).
Effect of N-Ethylmaleimide on the ATPase and DNA Helicase Activities of UL9C111A Protein-We predicted that cysteine 111 is responsible, at least in part, for the sensitivity of the UL9 protein DNA-dependent ATPase and DNA helicase activities to NEM. Consistent with our previous data (Table I, Fig. 3), the DNA-dependent ATPase and DNA helicase activities of the UL9 protein were inhibited by NEM modification (Fig. 7). In agreement with our prediction, NEM modification had no effect on the DNA-independent ATPase activity of UL9C111A protein and did not inhibit its DNA-dependent ATPase activity (Fig. 7,  A and B). Interestingly, NEM-modified UL9C111A protein actually exhibited an increased rate of DNA-dependent ATP hydrolysis (Fig. 7B). In contrast to the ATPase activities of UL9C111A protein, its DNA helicase activity, like that of UL9 protein, was inhibited by NEM modification (Fig. 7C).
Kinetic Characterization of UL9C111A Protein-To determine the basis of the defect in UL9C111A protein, a steadystate kinetic analysis of the DNA-dependent ATPase activity of UL9 and UL9C111A proteins was performed. Determination of k cat and K m ATP for the DNA-independent ATPase activity of UL9 and UL9C111A proteins was also attempted. However, rates of ATP hydrolysis failed to saturate with increasing ATP concentrations, exhibiting a linear relationship up to 10 mM ATP, making it impossible to determine k cat and K m ATP (data not shown). In contrast, the addition of increasing ATP concentrations to reactions containing UL9 or UL9C111A proteins and (dT) 60 cofactor resulted in typical Michaelis-Menten behavior (data not shown). The kinetic parameters for the DNA-dependent ATPase activity of UL9 and UL9C111A proteins are shown in Table II. The values for K m ATP and K m ssDNA are in agreement with those previously reported for UL9 protein (22). Consistent with the data shown in Fig. 5, k cat of UL9C111A protein is 2-fold lower than that of UL9 protein.
Interestingly, the defect in UL9C111A protein does not appear to be in its interaction with ssDNA cofactor, since it actually possess a lower K m ssDNA than UL9 protein. Consequently, the k cat /K m ssDNA ratios of UL9 and UL9C111A proteins are not significantly different. However, UL9C111A protein exhibits a 3-fold higher K m for ATP than UL9 protein. Accordingly, the k cat /K m ATP ratio of UL9C111A protein is almost 7-fold lower than that of UL9 protein.
Effect of the UL9C111A Mutation on Origin-dependent DNA Replication-The ability of UL9C111A protein to support origindependent DNA replication in vivo was determined by detecting DpnI-resistant oriS-containing pGEM822 in Southern blots. Fig. 8 shows a representative result of such an experiment. While replicated, DpnI-resistant pGEM822 was detectable in cells infected with A. californica NPV encoding wildtype UL9, no detectable DNA replication was observed in cells infected with A. californica NPV encoding UL9C111A or in mock-infected cells. The defect in origin-dependent DNA replication was not due to the lack of expression of UL9C111A protein, since cells used for determining DNA replication ac-tivity exhibited comparable expression of UL9 and UL9C111A proteins (data not shown).

DISCUSSION
In this paper, we have examined the importance of cysteine residues for the functions of the HSV-1 UL9 protein and described the properties of a mutant UL9 protein that bears a cysteine to alanine substitution at position 111.
The data show that the DNA helicase and DNA-dependent ATPase activities of the UL9 protein were stimulated by dithiothreitol, indicating the requirement for reduced cysteines (sulfhydryl groups). In addition, both activities were inactivated by modification with NEM. However, NEM modification of the UL9 protein did not affect its DNA-independent ATPase  7. Effects of N- and ssDNA-and origin-binding activities. These findings indicate that NEM modification does not indiscriminately inactivate the activities of the UL9 protein and therefore does not result in any gross structural alterations. Preincubation of the UL9 protein with ssDNA protected it from NEM inactivation presumably by masking crucial cysteine(s). Lower levels of protection were also provided by preincubation of UL9 protein with ATP and duplex DNA. These data indicate that the susceptible cysteine(s) is primarily associated with the ssDNAbinding site although they do not participate in ssDNA-binding directly, since NEM-modified UL9 protein retains ssDNA-binding activity. Because NEM modified the majority of cysteines in the UL9 protein and given that the majority of these were also protected from NEM modification by ssDNA, it was impossible to utilize this approach to identify the crucial cysteine(s). However, the cysteine(s) must be located in the N-terminal twothirds of the UL9 protein, since this domain retains ssDNAbinding, ATPase, and DNA helicase activities (23). Consequently, we employed site-directed mutagenesis to identify the pertinent residue. Cysteine 111 is one of four cysteines that are conserved in the N-terminal domain of herpesvirus origin-binding proteins. The proximity of cysteine 111 to the "Walker" type A adenine nucleotide-binding site and DNA helicase motif I (31)(32)(33) suggested to us that it may be responsible for NEMinduced inactivation of the DNA-dependent ATPase and DNA helicase activities of UL9 protein.
Our results show that cysteine 111 is an essential residue, since the mutant UL9C111A protein failed to support origindependent DNA replication in vivo. Substitution of cysteine 111 with alanine did not affect the DNA-independent ATPase activity of UL9 protein, suggesting that this residue is not directly involved in ATP binding or hydrolysis. This finding is consistent with the observation that NEM modification did not affect the DNA-independent ATPase activity of UL9 protein. Furthermore, the fact that UL9C111A protein retains an unaltered level of DNA-independent ATPase activity indicates that substitution of cysteine 111 with alanine did not lead to any gross structural alterations that may have impaired its catalytic activity. This conclusion is substantiated by the finding that UL9C111A protein retains wild-type levels of origin binding activity and also exhibits similar thermal stability to UL9 protein.
UL9C111A protein retains DNA-dependent ATPase activity albeit with a 2-fold lower k cat than UL9 protein. The ability of ssDNA to stimulate ATP hydrolysis in UL9C111A protein indicates that it has retained its ability to interact with ssDNA. This conclusion is supported by the finding that UL9C111A protein exhibits a K m for ssDNA that is actually slightly lower than that of UL9 protein. Consequently, the defect in UL9C111A protein is not in its interaction with ssDNA. Substitution of cysteine 111 with alanine also resulted in a 2-fold decrease in the specific activity of DNA unwinding.
The observation that rates of ATP hydrolysis did not saturate with increasing ATP concentrations unless ssDNA cofactor was present suggests that ssDNA binding increases the affinity of UL9 protein for ATP, possibly by inducing a conformational change. We propose that the 3-fold increase in K m for ATP of UL9C111A protein is due to the failure of ssDNAbinding to induce high affinity ATP binding, indicating that cysteine 111 is involved in coupling ssDNA-binding to ATP binding and subsequent hydrolysis. Moreover, the decrease in k cat and increase in K m ATP of UL9C111A protein, as manifested by a 7-fold lower k cat /K m ATP ratio, are responsible for the inability of UL9C111A protein to support origin-dependent DNA replication in vivo. Thus, at a K m of almost 2 mM ATP and at a reduced k cat , mutant UL9C111A protein is incapable of performing its essential role in viral origin-dependent DNA replication, presumably due to a defect at the level of DNA unwinding.
We targeted cysteine 111 in the hope of identifying the residue responsible for NEM-induced inactivation of UL9 protein. Consistent with our prediction, the DNA-dependent ATPase activity of UL9C111A protein is NEM-resistant. In fact, NEMmodified UL9C111A protein exhibited elevated DNA-dependent ATPase activity. This phenomenon may be the consequence of some structural alteration analogous to that seen with UL9DM27 protein, which lacks the C-terminal 27 amino acids of UL9 protein (29). Interestingly, while the DNA-dependent ATPase activity of UL9C111A protein is NEM-resistant, its DNA helicase activity, like that of UL9 protein, is NEM-sensitive. Accordingly, sensitivity of the UL9 protein DNA-dependent ATPase and DNA helicase activities to NEM is due to modification of at least two functionally important cysteines. The first, cysteine 111, is involved in coupling ssDNA binding to high affinity ATP binding. The second, presumably one of the three remaining conserved cysteines in the N-terminal domain of UL9 protein, is involved in coupling ATP hydrolysis to DNA unwinding.
In summary, given that NEM-modified UL9 protein lacks DNA helicase and DNA-dependent ATPase activities but retains DNA-independent ATPase and ssDNA-binding activities, we propose that cysteines are not required for catalysis or substrate binding but rather for signal transduction between ssDNA binding and ATP hydrolysis and for movement of the UL9 protein along DNA. It is possible that the critical cysteines are part of a channel that contains the ATP-and ssDNAbinding sites and other elements involved in translocation of UL9 protein along DNA. The presence of a "groove" that involves the ATP-and ssDNA-binding sites has been inferred from the crystal structures of the RecA protein as well as the PcrA and Rep DNA helicases (40 -42). Presumably, the function of such a groove is to transduce effects in response to ATP binding and/or hydrolysis that would enable the enzyme to translocate along DNA. We have identified cysteine 111 as an essential residue that is involved in coupling ssDNA binding to ATP binding and hydrolysis. In addition, cysteine 111 is one of at least two cysteines that are responsible for the sensitivity of the UL9 protein DNA-dependent ATPase and DNA helicase activities to NEM. Cysteines involved in metal binding in the E. coli PriA protein and HSV-1 UL52 DNA helicase-primase subunit have previously been implicated in DNA-dependent ATP hydrolysis and DNA unwinding (43,44). Our results are the first to show that a cysteine not involved in metal binding participates in the mechanism of DNA unwinding, specifically coupling ssDNA-binding to ATP-binding and hydrolysis.