©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Primase Active Site of the Herpes Simplex Virus Type 1 Helicase-Primase (*)

Stella Dracheva (1), Eugene V. Koonin (2), James J. Crute (1)(§)

From the (1) Department of Immunological Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368 and the (2) National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase composed of the products of the three DNA replication-specific genes UL5, UL8, and UL52 (Crute, J. J., and Lehman, I. R.(1991) J. Biol. Chem. 266, 4484-4488). The UL5 and UL52 products constitute a heterodimeric subassembly of the holoenzyme that contains both helicase and primase activities (Calder, J. M., and Stow, N. D.(1990) Nucleic Acids Res. 18, 3573-3578; Dodson, M. S., and Lehman, I. R.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1105-1109). The role of the UL52 product in the active HSV-1 helicase-primase was examined. A sequence located between residues 610 and 636 on the UL52 protein was found to be conserved among the UL52 homologues of eight herpesviruses. The carboxyl-terminal portion of this conserved sequence consisted of two Asp residues separated by a variable hydrophobic amino acid residue and is analogous to the divalent metal-binding site of DNA polymerases and several DNA primases. This motif has been designated the herpesvirus primase DXD motif. To study the role of the HSV-1 primase DXD motif in primase action, three site-directed changes were introduced into the UL52 gene. The helicase activity of the recombinant holoenzymes was unaffected by any of the introduced changes. Changing either of the two Asp residues that constitute the divalent metal-binding site (Asp or Asp) to Ala dramatically reduced the primase activity of the HSV-1 helicase-primase holoenzyme in vitro, whereas alteration of the nearby conserved residue Asn to Gly had minimal effect. Therefore, in the three-subunit HSV-1 helicase-primase, the UL52 product provides at least a part of the primase catalytic site.


INTRODUCTION

Herpes simplex virus type 1 (HSV-1)() contains seven nondispensable DNA replication-specific genes (1) . These seven genes encode four functionally different DNA replication proteins or protein complexes. A homodimeric origin-binding protein helicase is encoded by UL9, a monomeric SSB is encoded by UL29, and a heterodimeric DNA polymerase is encoded by UL30 and UL42. The products of the remaining three DNA replication genes, UL5, UL8, and UL52, constitute a heterotrimeric helicase-primase. The UL5 and UL52 products have been shown to form a stable isolable subassembly of the HSV-1 helicase-primase (2, 3) . This complex contains all of the measurable enzymatic activities associated with the holoenzyme. The UL8 product does not seem to participate directly in the catalysis of either DNA unwinding or RNA primer synthesis. Isolation of the individual UL5 and UL52 products has shown that neither protein alone has appreciable helicase activity; preparations of the UL52 protein have been found to contain low levels of primase activity.()

The predicted amino acid sequence of the UL5 product has been found to contain seven conserved amino acid sequence motifs typical of superfamily I of confirmed or putative helicases (4) . This suggests that the UL5 product functionally contributes to the helicase activity of the holoenzyme. The predicted amino acid sequence of the UL52 protein is not homologous to any known gene family.() We have now examined the function of the UL52 product in the active HSV-1 helicase-primase by a combination of sequence comparisons and site-directed mutagenesis. Multiple sequence alignment of the predicted amino acid sequence of the UL52 product with seven other identified herpesvirus UL52 homologues delineated several conserved regions. One of these resembled the putative metal-binding site found in prokaryotic and eukaryotic primases and eukaryotic DNA polymerases. This conserved region contained two Asp residues and was designated the herpesvirus primase DXD motif. To explore the function that this site provides in the HSV-1 helicase-primase, we introduced three point mutations into the DXD motif of the UL52 sequence. Recombinant helicase-primase holoenzymes were then expressed in insect cells, isolated, and studied in vitro by comparison to the wild-type enzyme. From this analysis, we have tentatively concluded that the UL52 product contains at least part of the primase catalytic site in the HSV-1 helicase-primase holoenzyme.


MATERIALS AND METHODS

Reagents

All reagents were of the highest quality available. Linearized baculovirus DNA and the baculovirus transfer vector pVL1393 were from Invitrogen; BaculoGold DNA was from Pharmingen. Grace's medium was obtained in powdered form from JRH Biosciences. Pluronic F-68 was from Life Technologies, Inc. Sf9 cells were from the American Type Culture Collection; Sf21 cells were from Invitrogen. Source 15Q was obtained from Pharmacia Biotech Inc. in bulk form and packed as an 8-ml column (1.0 10 cm) using a fast protein liquid chromatography system. A prepacked column of Sephacryl S-300 HR (1.6 60 cm; Pharmacia Biotech Inc.) was used for gel filtration. The plasmid pBluescript II SK was from Stratagene. Modified T7 DNA polymerase (Sequenase) was from United States Biochemical Corp. X174 single-stranded DNA was from New England Biolabs Inc. Radiolabeled reagents were from DuPont NEN. Oligonucleotides used as size standards were from Pharmacia Biotech Inc.

Amino Acid Sequence Analysis

Amino acid sequences were obtained from the GenBank Data Bank (Release 74.0). Data base searches for sequence similarity were performed using programs based on the BLAST algorithm (5) and the BLOSUM62 matrix for comparison of amino acid residues (6) . Multiple alignments of amino acid sequences were generated using the MACAW programs (7) . Data base searching for sequence motifs was performed using the programs DBSITE and FPAT (National Center for Biotechnology Information, National Institutes of Health).

Buffers

Buffer A was 20 mM NaHEPES, pH 7.5, 1.0 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 mM sodium bisulfite, pH 7.6, and 1.0 mM phenylmethylsulfonyl fluoride. Buffer B was Buffer A to which was added 10% (v/v) glycerol, 200 mM NaCl, and 2.0 M ammonium sulfate; sodium bisulfite was omitted. Buffer C was Buffer A to which was added 10% (v/v) glycerol, 1.0 mM EDTA, and 1.0 mM EGTA with sodium bisulfite deleted. Buffer D was 20 mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, 1.0 mM dithiothreitol, 5.0 mM MgCl, and 200 µg/ml acylated bovine serum albumin. Buffers were filtered through 0.22-µm membranes and degassed by sonication prior to use.

Cells, Viruses, and DNA

Routine methods were used throughout for the production of DNA constructs and recombinant baculoviruses (8, 9) . Spodoptera frugiperda cells (Sf9 or Sf21) were maintained in suspension culture at 27 °C with shaking in Grace's medium supplemented with 0.33% TC Yeastolate, 0.33% lactalbumin hydrolysate, 0.10% Pluronic F-68, and 10% heat-inactivated fetal bovine serum (10) . When Sf21 cultures were scaled to the 1-liter level, gentamicin sulfate (50 µg/ml) was added. The recombinant baculoviruses AcMNPV/UL5, AcMNPV/UL8, AcMNPV/UL30, and AcMNPV/UL52 (11, 12) were from Drs. Mark Dodson (University of Arizona), Thomas Hernandez (Stanford University), and Robert Lehman (Stanford University). AcMNPV/UL29 was constructed as follows. The RsrII-EarI fragment between HSV-1 coordinates 62069 and 58259 (13) was removed from the UL29-containing plasmid p8-BS (provided by Dr. David Knipe, Harvard University) and inserted into the transfer vector pVL1393 utilizing EcoRI linkers to yield pVL1393/UL29. The correct insert was confirmed by nucleotide sequencing across the start site of the transfer vector. AcMNPV/UL29 was isolated by several rounds of plaque purification from the virus stock generated by cotransfection of pVL1393/UL29 with linearized baculovirus DNA into Sf9 cells. AcMNPV/UL42 was constructed as follows. Uracil-rich single-stranded DNA was generated from the plasmid pNN4 (provided by Dr. Paul Olivo (Washington University) and Dr. Mark Challberg (National Institutes of Health)) in Escherichia coli strain CJ236 by infection with M13KO7 (14) . An additional BamHI restriction site was introduced by oligonucleotide-directed mutagenesis at HSV-1 coordinate 92094 (13) . This was termed pNN4/Bam. The fragment from the introduced BamHI site to the unique FspI site in the UL42 open reading frame was removed from pNN4/Bam, assembled with the balance of the UL42 open reading frame removed from pNN4, and ligated into BamHI-EcoRI-digested pBluescript II SK to create pBS2/UL42. The full-length UL42 open reading frame contained in the BamHI-EcoRI fragment was removed and inserted into pVL1393 to construct pVL1393/UL42. The identity of the construct was confirmed by nucleotide sequencing. The recombinant baculovirus AcMNPV/UL42 was obtained as described for AcMNPV/UL29. Virus stocks used for the production of recombinant protein were generated in Sf21 cells, and their titer was determined by plaque assay on Sf9 cells.

Construction of Expression Vectors for Altered UL52 Genes

Three site-directed mutations were introduced into the UL52 open reading frame. The UL52 coding region was removed from pVL941/UL52 (provided by Drs. Mark Dodson and Robert Lehman) by BamHI digestion and inserted into pBluescript II SK to create pBS2/UL52. Uracil-rich single-stranded DNA was obtained as described for pNN4 and used as template in three separate oligonucleotide-directed mutagenesis reactions. These changed UL52 codons for Asn, Asp, and Asp to Gly, Ala, and Ala, respectively. These constructs were termed pBS2/UL52(N624G), pBS2/UL52(D628A), and pBS2/UL52(D630A), respectively. The 540-base pair NcoI-NcoI fragment was then removed from each of the three constructs and reinserted into the parent pBS2/UL52 construct lacking the identical fragment to give the constructs pUL52(N624G), pUL52(D628A), and pUL52(D630A), respectively. The full-length altered UL52 open reading frames were then transferred to pVL1393 by BamHI digestion to create pVL1393/UL52(N624G), pVL1393/UL52(D628A), and pVL1393/UL52(D630A), respectively. The identity of each construct was then confirmed by nucleotide sequencing. The recombinant baculoviruses AcMNPV/UL52(N264G), AcMNPV/UL52(D628A), AcMNPV/UL52(D630A) were then obtained by cotransfection with the respective transfer plasmids as described for the isolation of AcMNPV/UL29, except that BaculoGold DNA was substituted for the linearized baculovirus DNA.

Expression of Wild-type and Altered HSV-1 Helicase-Primase Holoenzymes

Sf21 cells (3.0 liter) were grown to a density of 0.8-1.0 10 cells/ml, collected by centrifugation, and infected by resuspension in 0.1 culture volume of a viral inoculum calculated to yield a multiplicity of infection of 5-10 plaque-forming units of the indicated viruses/cell. When necessary, the volume was adjusted with the medium used for cell growth. The HSV-1 helicase-primases were expressed by triply infecting the Sf21 cells with AcMNPV/UL5, AcMNPV/UL8, and AcMNPV/UL52, AcMNPV/UL52(N624G), AcMNPV/UL52(D628A), or AcMNPV/UL52(D630A) to obtain the HSV-1 helicase-primase, HSV-1 helicase-primase [UL52(N624G)], HSV-1 helicase-primase [UL52(D628A)], or HSV-1 helicase-primase [UL52(D630A)], respectively. After allowing 1 h for viral adsorption, the cells were diluted to their original density with fresh medium, and protein expression was allowed to proceed for 60-68 h.

Preparation of Cell Extracts and Ammonium Sulfate Precipitation of HSV-1 Helicase-Primases

All procedures were performed at 4 °C unless indicated. The baculovirus-infected Sf21 cells were harvested by centrifugation, washed once with Grace's medium (9) , and resuspended in 7 volumes of Buffer A. ATP and MgCl were added to concentrations of 1.0 and 2.0 mM, respectively. Typical yields of cells were 7.0-10 g/liter. After incubation on ice for 10 min, the cells were lysed by Dounce homogenization (15-20 strokes with a tight fitting pestle), and the nuclear fraction was separated from the cytosolic fraction by centrifugation (1000 g, 15 min). Cytosolic fractions were retained and further clarified by ultracentrifugation for 75 min at 100,000 g. HSV-1 helicase-primases were precipitated with ammonium sulfate by mixing 1 volume of the postmicrosomal supernatant solution with 1 volume of Buffer B and incubating for at least 2 h on ice. The precipitates were harvested by centrifugation and resuspended in a minimal volume of Buffer C containing 200 mM NaCl. The conductivities of the samples were determined, and the samples were diluted with Buffer C to a conductivity corresponding to Buffer C containing 100 mM NaCl. The diluted extracts were then clarified by ultracentrifugation (440,000 g, 10 min).

Additional Purification of HSV-1 Helicase-Primases

The HSV-1 helicase-primases were purified from ammonium sulfate-precipitated cytosolic extracts by chromatography through Source 15Q and gel filtration through Sephacryl S-300 HR. The diluted clarified fraction was loaded onto the Source 15Q column equilibrated in Buffer C containing 100 mM NaCl; the column was washed with 1.5 column volumes of the equilibration buffer; and the protein was eluted with an 8-column volume linear gradient between Buffer C containing 100 mM NaCl and Buffer C containing 800 mM added NaCl. Fractions containing the HSV-1 helicase-primase eluted at a point in the gradient corresponding to 250 mM NaCl and were identified by the presence of DNA-dependent ATPase activity and the concomitant elution of the three polypeptides (M 120,000, 97,000, and 70,000) that form the HSV-1 helicase-primase holoenzyme. Active fractions were further fractionated by gel filtration through Sephacryl S-300 HR equilibrated in Buffer C containing 300 mM added NaCl. The HSV-1 helicase-primase eluted at a point in the chromatogram corresponding to ferritin (M 440,000). Protein concentrations of pooled fractions were determined utilizing a molar extinction coefficient at 280 nm of 269,000 M cm(15) . The enzyme was frozen in aliquots under liquid nitrogen and stored at -80 °C. Between 15 and 25 mg of nearly homogeneous enzyme was obtained from each preparation at a concentration of at least 1 mg/ml.

Isolation of the HSV-1 SSB and HSV-1 DNA Polymerase Holoenzyme

For the HSV-1 SSB, the baculovirus AcMNPV/UL29 was used to infect 6.0 liters of Sf21 cells. The isolation procedure was identical to that for the HSV-1 helicase-primase holoenzyme, except that ATP and MgCl were not added at the cell lysis step and the Source 15Q column volume was increased to 18 ml (1.6 9.0 cm). Two gel filtration runs were performed to accommodate the increased preparation size. For isolation of the HSV-1 DNA polymerase holoenzyme, Sf21 cells (6.0 liters) were doubly infected with AcMNPV/UL30 and AcMNPV/UL42. Lysates were prepared as described for the HSV-1 SSB. The enzyme was initially precipitated with Buffer B containing 2.5 M ammonium sulfate. The HSV-1 DNA polymerase was isolated as indicated for the HSV-1 SSB. Purified protein samples were quantitated with molar extinction coefficients at 280 nm of 70,400 and 114,000 M cm for the HSV-1 SSB and HSV-1 DNA polymerase holoenzyme, respectively (15) . 440 mg of nearly homogeneous HSV-1 SSB (10-15 mg/ml) and 20 mg of 95% pure HSV-1 DNA polymerase holoenzyme (1.2 mg/ml) were obtained.

DNA Helicase Assays

DNA helicase assays were performed with minor modifications as described using the double-tailed substrate (16). The [-P]ATP used to radiolabel the 68-mer oligonucleotide was changed to [-P]ATP. After gel filtration, the helicase substrate was concentrated by membrane diafiltration. Additionally, the concentration of the helicase substrate used in the unwinding reaction was increased to 50 µM (in nucleotide). Quantitation of the individual helicase reactions was as described (17) .

Coupled DNA Primase-DNA Polymerase Assays

Coupled DNA primase-DNA polymerase assays were performed essentially as described (18, 19). Primase-dependent DNA polymerase activity was assayed with the HSV-1 helicase-primase holoenzymes using either modified T7 DNA polymerase (Sequenase) or the HSV-1 DNA polymerase holoenzyme as the accessory DNA polymerase. Assays (25 µl) were assembled on ice in Buffer D with 1.0 µg of X174 single-stranded DNA, 5.8 pmol of HSV-1 SSB, 1.0 mM ATP, 1.0 mM GTP, 100 µM CTP, and 100 µM UTP. The three deoxynucleoside triphosphates dTTP, dCTP, and dGTP were added at a final concentration of 50 µM. [H]dATP was included at 20 µM at a final specific activity of 10 Ci/mmol. The indicated DNA polymerase was added (3.0 units of modified T7 DNA polymerase (Sequenase) or 2.7 pmol of HSV-1 DNA polymerase holoenzyme), and the reaction was initiated by the addition of 3.4 pmol of the indicated HSV-1 helicase-primase holoenzyme. One unit of DNA primase activity resulted in the incorporation of 1.0 pmol of dATP into polymeric DNA/h at 34 °C.

The native size and distribution of the products synthesized in the coupled assay were also visualized. Reactions were performed as described above for the radiolabeled coupled assay, except that [H]dATP was omitted and 50 µM dATP was included. Reactions were terminated by the addition of EDTA to 10 mM, phenol/chloroform/isoamyl alcohol (24:24:1)-extracted, ethanol-precipitated, resuspended in 10 µl of a glycerol-containing loading buffer, and electrophoresed through a 0.8% agarose gel. The products of the coupled DNA primase-DNA polymerase reaction were visualized by UV transillumination after staining with ethidium bromide.

Direct DNA Primase Assays

Direct primase assays were performed as described previously (18, 19, 20) . Reactions (25 µl) were assembled as described for the coupled assay with the omission of DNA polymerase and dNTPs. To visualize the products generated during the course of the primer synthesis reaction, 10 µCi of [-P]UTP was included in addition to the other three NTPs. Primer synthesis reactions were initiated by the addition of 3.4 pmol of wild-type or mutant HSV-1 helicase-primase holoenzyme. After incubation for 1.0 h at 34 °C, reactions were terminated by the addition of formamide to 50% (v/v) and heating to 95 °C. Products of the primase reaction were separated from the reactants by electrophoresis through 18% polyacrylamide gels containing 7.0 M urea as described (8) . Size standards of known chain length were radiolabeled with T4 polynucleotide kinase and [-P]ATP. These were included in parallel lanes as reference markers. After electrophoresis, the gel was fixed with 10% trichloroacetic acid and dried under vacuum, and the products of the primase reaction were visualized by autoradiography. The ability of the HSV-1 DNA polymerase to utilize the RNA primers synthesized in the direct primase reaction was also analyzed. For these experiments, the assay components were assembled as described for the direct gel assay. After the primer synthesis reaction, 2.7 pmol of HSV-1 DNA polymerase holoenzyme and a 50 µM concentration of each of the four dNTPs were added, and the reaction was allowed to proceed for an additional 1.0 h. The products of the assay were processed as described for the direct primase assay.


RESULTS

Identification of the Herpesvirus Primase DXD Motif

Amino acid and nucleotide sequence data base searches with the predicted HSV-1 UL52 amino acid sequence revealed significant similarity (probability of matching by chance alone below 10) only to the homologous proteins of other herpesviruses. Alignment of the predicted amino acid sequences of eight herpesvirus UL52 homologues showed several conserved blocks. Particularly noticeable was a conserved region of variable hydrophobic-hydrophilic character contained within the UL52 homologues. This region corresponded to residues 607-634 on the predicted UL52 amino acid sequence (Fig. 1). The carboxyl-terminal one-third of this region contained two conserved Asp residues (UL52 Asp and Asp) within a local group of hydrophobic amino acid residues. This structure, designated as the herpesvirus primase DXD motif, is similar to part of the active center of family B DNA polymerases (e.g. region I) and also to one of the conserved motifs found in prokaryotic and eukaryotic primases (21, 22, 23) .


Figure 1: Alignment of the putative metal-binding sites in primases, DNA polymerases, and UL52-related proteins: identification of the herpesvirus primase DXD motif. Multiple sequence alignments of the four groups of proteins were generated by the MACAW program, and the conserved motifs were superimposed manually. A, prokaryotic DNA primases; B, eukaryotic primases; C, DNA-dependent DNA polymerases and terminal deoxynucleotidyltransferases; D, herpesvirus UL52 homologues. A-C depict the short amino acid residue stretches containing the ``DXD'' motif; D shows the complete block delineated by the MACAW program for the herpesvirus UL52 homologues. The consensus sequence, derived separately for each group, consists of amino acid residues that are conserved in all sequences (upper-case letters) or in all but one sequence (lower-case letters). Consensus abbreviations are as follows: U, bulky aliphatic residue (I, L, V, or M); &, bulky hydrophobic residue (I, L, V, M, F, Y, or W); and ., any residue. The two conserved aspartic acid residues that may directly interact with the metal cation are in boldface type. For each protein, the position of the first aligned residue in the sequence is indicated; the numbers in parentheses are for partial sequences. Each sequence is accompanied by the SwissProt, PIR, or GenBank accession number. B.subt., Bacillus subtilis; B.aphi., Buchnera aphidicola; R.prow., Rickettsia prowazekii; POL-beta, polymerase ; TDT, terminal deoxynucleotidyltransferase; VZV, varicella-zoster virus; HCMV, human cytomegalovirus; MCMV, murine cytomegalovirus; HHV6, human herpesvirus 6; EHV, equine herpesvirus; EBV, Epstein-Barr virus; HVS, Herpesvirus saimiri.



Production of UL52-mutated HSV-1 Helicase-Primase Holoenzymes

We engineered three different alterations in the HSV-1 UL52 sequence such that the UL52 protein was changed in the domain that contains the HSV-1 primase DXD motif. Residues Asp and Asp were changed to Ala, and the nearby conserved residue Asn was changed to Gly. Recombinant baculoviruses were then constructed (AcMNPV/UL52(D628A), AcMNPV/UL52(D630A), and AcMNPV/UL52(N624G)) that expressed the UL52 protein with the respective alterations. Altered HSV-1 helicase-primases were individually expressed in insect cells triply infected with baculoviruses recombinant for wild-type UL5, wild-type UL8, and one of the altered UL52 genes. The HSV-1 helicase-primases [UL52(N624G)], [UL52(D628A)], and [UL52(D630A)] were isolated according to a rapid purification scheme, and their in vitro properties were determined and compared with identically prepared wild-type HSV-1 helicase-primase.

Helicase Activity Is Normal in the UL52-mutated HSV-1 Helicase-Primase Holoenzymes

The helicase activity of the HSV-1 helicase-primase holoenzyme was compared with that of the enzymes containing the UL52 product mutations UL52(D628A), UL52(D630A), and UL52(N624G). When the individual enzymes were assayed for associated helicase activity, the three altered helicase-primase holoenzymes were found to contain helicase activity nearly identical to the wild-type HSV-1 helicase-primase (Fig. 2). The differences observed in helicase activity were 15% of the wild-type enzyme activity.


Figure 2: Helicase activity of the HSV-1 helicase-primase holoenzyme is unaffected by mutations introduced into the UL52 subunit. Helicase activities associated with the wild-type and UL52-mutated HSV-1 helicase-primase holoenzymes were analyzed as described under ``Materials and Methods.'' A, lanes 1-5, 6-10, 11-15, and 16-20 represent groups of helicase assays performed with the HSV-1 helicase-primase holoenzymes containing wild-type UL52, UL52(D628A), UL52(D630A), or UL52(N624G), respectively. Each set of five assays was assembled to contain increasing amounts of the indicated HSV-1 helicase-primase holoenzyme: 0.0, 1.7, 3.4, or 5.1 pmol. The final lane of each group contained 5.1 pmol of the indicated HSV-1 helicase-primase holoenzyme with the omission of ATP from the assay. The position of the electrophoretic origin of the gel (origin) and the point of migration of the dehybridized radiolabeled oligonucleotide product (68-mer) of the helicase reaction are indicated. B, shown is the quantitation of helicase activity. The activity of individual helicase reactions outlined in A was quantitated as described under ``Materials Methods.'' The helicase activity of the individual HSV-1 helicase-primase holoenzymes was as follows: , wild-type; D1, UL52(D628A); D2, UL52(D630A); N, UL52(N624G).



DNA Synthesis Initiated by the Primase of the HSV-1 Helicase-Primase Holoenzyme Requires an Intact Herpesvirus Primase DXD Motif

Two types of indirect DNA primase assays were performed with the wild-type and UL52-mutated HSV-1 helicase-primase holoenzymes. Each assay examined the coupling of the primase activity of the indicated HSV-1 helicase-primase holoenzyme to the DNA synthetic activity of a heterologous or homologous DNA polymerase (Sequenase or the HSV-1 DNA polymerase holoenzyme). In assays performed using a radiolabeled dNTP to monitor coupled DNA primase-DNA polymerase DNA synthesis, we found that the alteration of either Asp or Asp to Ala in the UL52 protein [UL52(D628A)] or [UL52(D630A)] reduced holoenzyme priming activity to near background levels (). Alteration of the conserved Asn in the UL52 product [UL52(N624G)] had a minimal effect on the priming activity of the isolated holoenzyme. Moreover, primase activity was independent of the DNA polymerase used in the coupled assay (compare values obtained with Sequenase with results obtained with the HSV-1 DNA polymerase holoenzyme).

The other type of indirect primase assay examined the native size and distribution of products synthesized on X174 single-stranded DNA (Fig. 3). Native agarose gel electrophoretic analysis of the products synthesized in the indirect primase assay showed that alteration of either Ala or Ala in the UL52 product [UL52(D628A)] or [UL52(630A)] reduced the coupled DNA synthetic activity of the holoenzyme to background levels. Minimal full-length replicative form DNA was generated in the synthesis reaction when Sequenase was used as the coupling DNA polymerase (Fig. 3, compare lanes2 and 3 with lane5). When the HSV-1 DNA polymerase was used to couple RNA primer synthesis to DNA chain elongation, slightly greater amounts of replicative form products were noted when compared with the addition of DNA polymerase alone (Fig. 3, compare lanes7 and 8 with lane10). Furthermore, analysis of the HSV-1 helicase-primase holoenzyme [UL52(N624G)] showed near wild-type priming levels with either DNA polymerase used (Fig. 3, compare lanes4 and 9 with lanes1 and 6).


Figure 3: DNA synthesis coupled to RNA priming by the HSV-1 helicase-primase holoenzyme requires an intact herpesvirus primase DXD motif. Coupled DNA primase-DNA polymerase assays were performed as described under ``Materials and Methods'' for the indirect agarose gel-based assay. Lanes 1-5, Sequenase was used as the coupled DNA polymerase; lanes 6-10, the HSV-1 DNA polymerase holoenzyme was used as the coupled DNA polymerase. The primase used in individual assays was as follows: lanes1 and 6, HSV-1 helicase-primase holoenzyme (wild-type); lanes2 and 7, HSV-1 helicase-primase holoenzyme [UL52(D628A)]; lanes3 and 8, HSV-1 helicase-primase holoenzyme [UL52(D630A)]; lanes4 and 9, HSV-1 helicase-primase holoenzyme [UL52(N624)]. Assays in lanes5 and 10 contained no added helicase-primase. Lanes M contained molecular size markers of known length in kilobase pairs (kbp). Lane ss contained the input single-stranded DNA substrate. Lane ds contained X174 replicative form I (faster migrating band) and II (slower migrating band) double-stranded DNAs to indicate the position of migration of the fully duplex product of the primase-polymerase reaction.



The HSV-1 Primase DXD Motif Is Essential for Primer Synthesis by the HSV-1 Helicase-Primase Holoenzyme

The size distribution and utilization of primers synthesized by wild-type and altered HSV-1 helicase-primase holoenzymes were examined directly. The HSV-1 helicase-primase holoenzyme and the HSV-1 helicase-primase [UL52(N624G)] both synthesized primers of 12 nucleotides in length (Fig. 4, lanes1 and 4, respectively). Some RNA products of shorter chain length were also seen. In addition, the autoradiograph showed approximately the same amount of total RNA synthesized by each enzyme (Fig. 4, compare lanes1 and 4). The full-length primers synthesized by either the HSV-1 helicase-primase holoenzyme or the HSV-1 helicase-primase holoenzyme [UL52(N624G)] were utilized when the HSV-1 DNA polymerase holoenzyme and the four dNTPs were included in the reaction (note transfer of radiolabeled material of 12 nucleotides in size to the upper part of the autoradiogram in Fig. 4 , lanes6 and 9). In contrast, the mutant HSV-1 helicase-primase holoenzyme [UL52(D628A)] or [UL52(D630A)] contained negligible RNA primer synthetic activity (Fig. 4, lanes2 and 3, respectively). By densitometric scanning (Fig. 4, lanes 1-5), the difference in activity between the primase-proficient (wild-type or [UL52(N624G)]-altered HSV-1 helicase-primase holoenzyme) and the primase-deficient (HSV-1 helicase-primase holoenzyme [UL52(D628A)] or [UL52(D630A)]) enzymes was estimated to be at least 100-fold.


Figure 4: The herpesvirus primase DXD motif is essential for the synthesis of RNA primers by the HSV-1 helicase-primase holoenzyme. Direct primase assays were performed as described under ``Materials and Methods.'' The primase used in the individual assays was as follows: lanes 1 and 6, HSV-1 helicase-primase holoenzyme (wild-type); lanes2 and 7, HSV-1 helicase-primase holoenzyme [UL52(D628A)]; lanes3 and 8, HSV-1 helicase-primase holoenzyme [UL52(D630A)]; lanes4 and 9, HSV-1 helicase-primase holoenzyme [UL52(N624G)]. Lanes5 and 10 contained no added helicase-primase. Assays analyzed in lanes6-10 included the HSV-1 DNA polymerase holoenzyme and the four dNTPs. Origin, the electrophoretic origin of the gel; 20nt, 16nt, 12nt, and 10nt, the positions of reference oligonucleotides electrophoresed in parallel lanes.




DISCUSSION

We have focused our current studies on the primase of the HSV-1 helicase-primase enzyme complex. In performing multiple sequence alignments of UL52 homologues from eight different herpesviruses, we delineated several conserved regions. One of the regions contained two conserved Asp residues in the predicted HSV-1 UL52 amino acid sequence at positions 628 and 630. The two Asp residues were contained within a locally hydrophobic region that resembled the putative divalent metal-binding site identified in many DNA polymerases and primases. We have designated the conserved structure the herpesvirus primase DXD motif. Because of the similarity between the herpesvirus primase DXD motif and the DNA polymerase metal-binding site, in addition to the presence of this motif on other identified primases, the role of this structure in the HSV-1 helicase-primase holoenzyme was explored. Specific changes were introduced into the UL52 sequence by site-directed mutagenesis, and the resultant HSV-1 helicase-primase holoenzymes were analyzed.

In helicase assays performed on the altered HSV-1 helicase-primase holoenzyme [UL52(D628A)], [UL52(D630A)], or [UL52(N624G)], ATP-dependent unwinding was nearly the same as in the wild-type enzyme. The slight differences observed may reflect variability in enzyme preparations rather than intrinsic changes in enzyme activity. More interestingly, profound differences were observed in the ability of the enzymes to synthesize RNA primers. In either indirect or direct primase assays, alteration of either of the Asp residues in the HSV-1 primase DXD motif (Asp or Asp) dramatically reduced the in vitro DNA priming activity of the HSV-1 helicase-primase holoenzyme. From the gel-based assay that directly examined RNA primer synthesis by the HSV-1 helicase-primases, the reduction in primase activity is estimated to be 100-fold. The HSV-1 helicase-primase [UL52(N624G)] functioned similarly to the wild-type enzyme. This implies that despite a primary amino acid sequence proximity, the conserved Asn does not participate directly in mediating RNA primer synthesis.

Therefore, at least a part of the primase active site of the HSV-1 helicase-primase is contained within the UL52 product of the heterotrimeric holoenzyme. The two Asp residues contained within the HSV-1 primase DXD motif on the UL52 protein were found to be essential for in vitro and presumably in vivo RNA primer synthesis. Using a strategy of mutating conserved charged residues in the UL52 protein, Klinedinst and Challberg (25) have also concluded that the UL52 product is the primase subunit of the HSV-1 helicase-primase holoenzyme. By analogy to the similar motif in conserved region I of the eukaryotic DNA polymerases (21, 22, 24) , we tentatively propose that the critical function of the herpesvirus primase DXD motif is to bind NTP by coordination to nucleotide-bound Mg. This NTP-binding site is unrelated to the NTPase site on the HSV-1 helicase-primase holoenzyme. Alteration of the UL5 NTP-binding motif results in enzyme devoid of NTPase and helicase activities with enhanced primase activity.() At this time, it is not known which additional structures on the UL52 protein mediate other functions involved in RNA primer synthesis. Other studied DNA polymerases and primases have been found to contain all the structural domains necessary for nucleotide primer elongation on a single polypeptide chain. It is likely this may be the case with the UL52 subunit of the HSV-1 helicase-primase.

Primer synthesis may not be the only function provided by the HSV-1 UL52 protein in the helicase-primase. Although in the other studied helicase-primase systems subunits specific for primase and helicase activities have been delineated (e.g. T4 gene 41/61, T7 gp4/gp4*, and E. coli dnaB/dnaG helicase-primases) (26) , the herpesvirus helicase-primases have not been examined sufficiently for this conclusion to be drawn. Additionally, since activities involved in primase function, NTP interaction, and DNA binding are also required for DNA unwinding, functional overlap between RNA primer formation and duplex DNA unwinding may occur. Furthermore, ``zinc finger'' motifs are thought to be essential for the sequence-preferred priming activity demonstrated for several prokaryotic primases (27) . Interestingly, within the herpesvirus UL52 homologues, there also exists a conserved zinc finger domain at the extreme carboxyl-terminal part of the protein.() This structure may contribute to a mechanism that coordinates Okazaki strand RNA priming with duplex DNA unwinding at the advancing herpes replication fork. Ongoing studies are focused on delineating additional functional domains within the UL52 protein of the HSV-1 helicase-primase.

  
Table: Primase activity of wild-type and altered HSV-1 helicase-primase holoenzymes

Primase assays were performed as described under ``Materials and Methods'' for the indirect assay. Assays were initiated by the addition of the indicated HSV-1 helicase-primase holoenzyme and incubated for 1.0 h. One unit of primase activity incorporates 1.0 pmol of dNTP into polymeric DNA/h. Background incorporation values of 20.2 and 5.4 pmol were obtained in the absence of added helicase-primase for Sequenase and the HSV-1 DNA polymerase holoenzyme, respectively. These were subtracted to obtain the results presented.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 203-791-6163; Fax: 203-791-6196.

The abbreviations used are: HSV-1, herpes simplex virus type 1; SSB, single-stranded DNA-binding protein; AcMNPV, Autographa californica nuclear polyhedrosis virus.

S. Goldrick, M. S. Dodson, and J. J. Crute, unpublished observations.

E. V. Koonin, unpublished observations.

S. Goldrick, S. Dracheva, and J. J. Crute, unpublished observations.

E. V. Koonin and J. J. Crute, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to many of our colleagues for aid during the course of this investigation. Drs. Mark Dodson, Thomas Hernandez, and Robert Lehman generously provided the HSV-1 UL5, UL8, UL30, and UL52 recombinant baculoviruses. Drs. Paul Olivo and Mark Challberg made available the HSV-1 UL42-containing plasmid pNN4 and Dr. David Knipe the HSV-1 UL29-containing plasmid p8-BS. Drs. Daniel Tenney and Robert Hamatake made available details of work in progress on the primase of the HSV-1 helicase-primase. We also wish to extend our appreciation to Susan Goldrick for constructing the UL42 and UL29 recombinant baculoviruses, maintaining viral stocks, and overexpressing several of the reagents used here. Additionally, we thank Dr. Eugene McNally and Paul McGoff for helpful suggestions used in the development of scaled procedures for the isolation the HSV-1 replication proteins and Dr. Robert Eckner for assembly of the helicase substrate.


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