©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence-dependent Primer Synthesis by the Herpes Simplex Virus Helicase-Primase Complex (*)

Daniel J. Tenney (§) , Amy K. Sheaffer , Warren W. Hurlburt , Marc Bifano , Robert K. Hamatake

From the (1) Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The herpes simplex virus helicase-primase complex, a heterotrimer of the UL5, UL8, and UL52 proteins, displays a single predominant site of primer synthesis on X174 virion DNA (Tenney, D. J., Hurlburt, W. W., Micheletti, P. M., Bifano, M., and Hamatake, R. K. (1994) J. Biol. Chem. 269, 5030-5035). This site was mapped and found to be deoxycytosine-rich, directing the synthesis of a primer initiating with several guanine residues. The size and sequence requirements for primer synthesis were determined using oligonucleotides containing variations of the predominant template. Although the efficiency of primer synthesis on oligonucleotides was influenced by template size, it was absolutely dependent on nucleotide sequence. Conversely, the ATPase activity on oligonucleotide templates was dependent on template size rather than nucleotide sequence. Furthermore, only oligonucleotides containing primase templates were inhibitory in a coupled primase-polymerase assay using X174 DNA as template, suggesting that primer synthesis or primase turnover is rate-limiting. Additionally, stimulation of helicase-primase by the UL8 component and that by the ICP8 protein were shown to differ mechanistically using different templates: the UL8 component stimulated the rate of primer synthesis on X174 virion DNA and oligonucleotide templates, while ICP8 stimulation occurred only on X174 virion DNA.


INTRODUCTION

Replication of herpes simplex virus type 1 (HSV)() DNA during infection utilizes viral proteins that display functions conserved throughout DNA replication systems. The viral proteins that are essential in vivo include a heterodimeric processive DNA polymerase, a heterotrimeric helicase-primase complex, a single-stranded DNA-binding protein, and an origin of DNA replication-binding protein (reviewed in Refs. 1 and 2). The helicase-primase complex, composed of the UL5, UL8, and UL52 proteins (UL5/8/52), possesses helicase, DNA-dependent NTPase, and primase activities (3, 4, 5) . By virtue of these activities and analogies with other systems, the helicase-primase complex is thought to unwind DNA at the replication fork (helicase) and to synthesize oligoribonucleotide primers for use by DNA polymerase (primase).

In vitro, a subassembly of the UL5 and UL52 proteins displays the activities of the heterotrimer (6, 7, 8) . However, aside from low levels of NTPase activity from the UL5 protein (9) ,() the individual subunits of the helicase-primase complex are inactive (6, 7, 9, 10) . This has made assignment of individual functions to the specific subunits difficult. Recent mutational analysis of the UL52 protein has been used to suggest its role in the primase rather than helicase activity of the UL5/52 subassembly (12, 13) . In addition, mutation of conserved helicase domains in UL5 eliminates virus replication (14) and abolishes low levels of NTPase activity of the isolated UL5 subunit.However, both UL5 and UL52 are required for helicase activity (6) .

The UL8 protein has been shown to have an auxiliary role in primase activity in vitro (9) , specifically in stimulating the level of primer synthesis (11) . UL8 also facilitates a functional interaction of helicase-primase with the HSV single-stranded DNA-binding protein (ICP8) in vitro, resulting in increased primer synthesis when the complex also contains UL8.() UL8 has also been implicated in directing nuclear localization of the heterotrimeric complex in vivo (15) and in the formation of a complex of helicase-primase with the origin-binding protein (16) . Furthermore, recent studies using an in vitro model of HSV rolling circle replication were used to demonstrate that UL8 is required for the synthesis of long leading strands of DNA.()

In general, template sequence requirements for primases are thought to be rather nonspecific to enable primers to be synthesized frequently during lagging strand DNA replication (reviewed in Ref. 17). On the other hand, the primases of Escherichia coli (18, 19, 20) , T4 (21) and T7 (22) , as well as the mammalian polymerase--primase complex (23) have all been shown to invoke some level of template specificity for their initiation. Here we identify the predominant HSV primase initiation site within the X174 virion DNA template and use DNA oligonucleotides to define the size and sequence requirements for primer synthesis. In contrast with primer synthesis, the ATPase activity of helicase-primase was found to be independent of nucleotide sequence. We also use oligonucleotide templates to further characterize the activity of the enzyme and to distinguish the stimulation of primer synthesis by the UL8 component of helicase-primase from that by HSV ICP8 single-stranded DNA-binding protein.


EXPERIMENTAL PROCEDURES

Nucleic Acids

Single-stranded X174 and M13 virion DNAs were from New England Biolabs Inc. and Life Technologies, Inc., respectively. X174 DNA sequence numbering was according to the GenBank/EMBL Data Bank (accession number J02482) (24) . Oligonucleotide DNAs were obtained from Genosys Biotechnologies, Inc. (The Woodlands, TX), except for those containing the HSV origin of replication (25) : ori1 oligonucleotide sequence, AGCTTGGGTAAAAGAAGT-GAGAACGCGAAGCGTTCGCACTTCGTCCCAATATATATATATTA-TTAGGGCGAAGTGCGAGCACTGGCGCCG; and ori2 oligonucleotide sequence, GATCCGGCGCCAGTGCTCGCACTTCGCCCTAATAATAT-ATATATATTGGGACGAAGTGCGAACGCTTCGCGTTCTCACTTCT-TTTACCC. These were similar to those used by Hazuda et al. (26) and were obtained from National Biosciences (Plymouth, MN). Polyadenosine, polyguanosine, polydeoxyadenosine, and polydeoxythymidine were obtained from Pharmacia Biotech Inc.

Expression and Purification of Recombinant Proteins

HSV proteins were expressed by recombinant baculovirus infection of Sf9 insect cells (Invitrogen). Baculoviruses expressing the HSV UL5, UL8, and UL52 proteins (27) and the UL30 protein (HSV DNA polymerase) (28) were a generous gift of I. Robert Lehman. The baculoviruses expressing the HSV UL42 (29) and ICP8 (11) proteins were described previously. Insect cells were cultured in spinner flasks with large micocarrier impellers (Bellco Glass, Inc.) containing serum-free Sf900 medium (Life Technologies, Inc.) with 0.1% (w/v) pluronic F-68 (Life Technologies, Inc.) and penicillin/streptomycin to 100 µg/ml.

Purification of the recombinant HSV DNA polymerase-UL42 complex and ICP8 (28) and of the UL5/52 subassembly and UL8 (11) was described previously. Protein concentrations were determined by absorbance at 280 nm using calculated extinction coefficients (30) based on predicted amino acid sequences.

Coupled Primase-Polymerase Assays

Primase-dependent HSV DNA polymerase activity was assayed as described (11) in reactions modified from those of Sherman et al. (9) . [H]dTTP (78 Ci/mmol; DuPont NEN) incorporation using 50 ng of X174 virion DNA template (28.6 fmol of circular molecules) was terminated by trichloroacetic acid precipitation as described (29) . HSV DNA polymerase-UL42, ICP8, UL5/52, and UL8 were added as indicated. Although not extensively characterized, HSV helicase-primase is most active in this assay, where 400-1000 molecules of the primase are required for the extension of a single primer by HSV DNA polymerase.()

Direct Oligoribonucleotide Primer Synthesis Assay

Primer synthesis assays were carried out as described (11) . 10-µl reactions contained X174 virion DNA at a 30 µg/ml nucleotide concentration (17 n M circular molecules) except where noted. Oligonucleotide DNAs were used at 2 µ M linear molecules. Products were resolved by electrophoresis on denaturing 7 M urea, 20% polyacrylamide, Tris borate/EDTA (TBE) gels and visualized by autoradiography.

Kinetic analysis of primer production was performed with various concentrations of 50-mer oligonucleotide template (see , o50) and 1.88 pmol of UL5/52 with or without 5.6 pmol of UL8. Autoradiographs were scanned using an LKB laser densitometer and analyzed using Gelscan XL software (Pharmacia Biotech Inc.). Vand Kvalues were determined using KinetAsyst II software (IntelliKinetics, Inc.), which fit the amount of primers synthesized ( v) to the following equation: v = VS/( K+ S). Vis expressed in absorbance units from scanning laser densitometry of autoradiographs, and Kfor the DNA template is expressed in micromolar nucleotide.

Mapping of the Predominant Primase Initiation Site

Following radiolabeled primer synthesis on X174 DNA, dNTPs were added to 50 µ M each along with MgClto 10 m M and 0.25 units of modified T7 DNA polymerase (Sequenase, United States Biochemical Corp.). Radiolabeled primers were extended during incubation at 37 °C for 60 min. The resultant double-stranded DNA molecules were digested with restriction enzymes and resolved by nondenaturing polyacrylamide gel electrophoresis (PAGE) using 4% polyacrylamide (19:1 acrylamide/bisacrylamide), TBE gels and by denaturing PAGE using 7 M urea, 8% acrylamide, TBE gels, followed by autoradiography. Size markers consisted of restriction enzyme-digested X174 replicative factor I (supercoiled) DNA that was end-labeled with [-P]ATP using T4 polynucleotide kinase.

DNA-dependent ATPase

Assays for DNA-dependent ATPase activity were modified from the method of Crute et al. (3) and contained 50 m M Tris-HCl (pH 8), 10% (w/v) glycerol, 2 m M ATP, 4 m M MgCl, 1 m M dithiothreitol, and 10-20 µg/ml M13 virion effector DNA in a 50-µl total volume. Incubation was for 30 min at 30 °C. The hydrolysis of ATP was determined by the release of inorganic phosphate (31) by adding 20 µl of the reaction to 300 µl of 0.03375% (w/v) malachite green, 0.3% ammonium molybdate. Absorbance at 620 nm was measured.


RESULTS

The Preferred HSV Primase Initiation Site in X174 Virion DNA

We reported previously that HSV helicase-primase synthesizes primers at predominant sites within X174 and M13 virion DNA templates (11) . To investigate the template specificity of the enzyme, we mapped the predominant site within X174 DNA. Radiolabeled primers were synthesized on X174 DNA with HSV helicase-primase. Incubation of the primed X174 molecules with DNA polymerase and dNTPs resulted in a completely double-stranded DNA molecule except for a nick or gap at the original site of primer initiation. The DNA was digested with restriction endonucleases, and the products were resolved by native PAGE (Fig. 1 A). The predominant radiolabeled primer was localized to the 770-base pair HincII fragment (nucleotides 2593-3362). Further digestions mapped the primer to the 270-base pair HincII- SacII fragment (nucleotides 2593-2862) and the 182-base pair HinfI- SacII fragment (nucleotides 2681-2862). This primer was synthesized at multiple concentrations of enzyme and in the presence or absence of the UL8 subunit (Fig. 1 A). Resolution of the primer-initiated product from the HincII digestion by denaturing PAGE revealed it to be 112 nucleotides in length, thus originating 112 nucleotides from the HincII site at nucleotide 2593, corresponding with a primer initiation at X174 nucleotide 2705 (Fig. 1 B). Although Fig. 1 B shows the total product from the HincII digestion, the purified radiolabeled 770-nucleotide product from nondenaturing PAGE was also 112 nucleotides in length on denaturing PAGE (data not shown). The template was found to be deoxycytosine-rich, directing the synthesis of a guanine-rich primer. The size of the predominant primer synthesized was previously estimated to be 12 nucleotides based on comparison with end-labeled poly(A), partially digested with ribonuclease U2 (11) . By comparison with similarly digested radiolabeled poly(G), however, we now surmise that the primer is 10 nucleotides in length (data not shown). This estimate is supported by the results presented below. Therefore, the sequence of the primer synthesized using the predominant template is 5`-GGGAGGGUAG-3`.


Figure 1: Mapping of the predominant HSV primase initiation site in X174 virion DNA. Double-stranded X174 virion DNA, radiolabeled by HSV helicase-primase, was digested with restriction enzymes and separated by electrophoresis (see ``Experimental Procedures''). A, nondenaturing 4% PAGE of restriction fragments of X174 DNA initiated with radiolabeled primers. Lanes 1-3 contained X174 DNA digested with HincII, lanes 4-7 with HincII and SacII, and lanes 8-11 with SacII and HinfI. The positions of end-labeled X174 HincII restriction fragments electrophoresed in parallel are indicated by hatch marks. Arrowheads indicate the 770-nucleotide HincII, 270-nucleotide HincII/ SacII, and 185-nucleotide SacII/ HinfI fragments. Reactions in lanes 4, 5, 8, and 9 contained 5 pmol of UL5/52, and those in lanes 6, 7, 10, and 11 contained 15 pmol of UL5/52. A 3-fold molar excess of UL8 was added to reactions in lanes 5, 7, 9, and 11. B, denaturing 7 M urea, 8% polyacrylamide gel of total products of HincII restriction digests as described for A ( lane 3). The predominant radiolabeled product of 112 nucleotides is indicated by the arrowhead. The size was determined by a sequencing ladder of defined DNAs run in parallel ( lanes 2-5).



HSV Primase Activity on Oligonucleotide Templates

Examination of the sequence surrounding X174 nucleotide 2705 revealed several areas of potential secondary structure (data not shown). By analogy with other primases (reviewed in Ref. 17), secondary structure could potentially facilitate initiation by HSV helicase-primase. We tested the ability of an oligonucleotide containing the sequence surrounding the predominant site but with little potential for secondary structure (, oligonucleotide o50) to serve as a template for primer synthesis. The results (Fig. 2 A) indicate that the oligonucleotide is able to serve as a template for the synthesis of the same size primer as that produced using the X174 virion DNA molecule using [-P]UTP labeling. Furthermore, the addition of the UL8 component to the UL5/52 subassembly increased the amount of primers synthesized on the oligonucleotide as well as the X174 circular molecule (compare - and + UL8 lanes). The two faster migrating bands of lower intensity were found by RNase sequencing to initiate with one or two G residues rather than three G residues of the 10-mer (data not shown). An oligonucleotide with the complementary sequence to o50 (, o50r) could not be used as a template (Fig. 2 A). These results confirm the mapping of the preferred site and suggest that nucleotide sequence and not secondary structure most likely determines recognition by the enzyme. Interestingly, oligonucleotides containing 84 nucleotides that comprise the entire HSV origin of replication sequence (ori1 and ori2; see ``Experimental Procedures'' for sequence), including deoxycytosine-rich regions similar to the mapped template in X174, also failed to serve as substrates for primer synthesis (Fig. 2 A).


Figure 2: Primer synthesis by the HSV helicase-primase complex on X174 virion DNA and oligonucleotide templates. Primer synthesis reactions were analyzed by 20% denaturing PAGE (see ``Experimental Procedures''). A, reactions contained 1.88 pmol of the UL5/52 subassembly without (-) or with (+) 5.6 pmol of UL8 added and labeling with [-P]UTP. The DNA template in each reaction is indicated above the lanes (see Table I): none, no DNA template; X, X174 virion DNA; o50, X174 nucleotides 2681-2730; o50r, complement of o50; ori1 and ori2 contain the HSV origin of replication sequence. The position of the predominant 10-mer oligoribonucleotide primer is indicated with an arrowhead. B, primer synthesis reactions including UL8 as described for A were labeled with [-P]UTP, [-P]CTP, or both on various DNA templates as indicated. -, no DNA.



The template activity of oligonucleotide o50 was confirmed further using labeling conditions with [-P]CTP instead of and in combination with [-P]UTP (Fig. 2 B). These results indicate that a DNA oligomer can serve as a template for the HSV primase and establish the importance of template sequence in initiation of primer synthesis.

Size and Sequence Requirements for the HSV Primase Template

The next series of experiments was designed to determine the influence of oligonucleotide template size and nucleotide sequence on primer synthesis. Fig. 3 A shows the result of truncating the template at the 3`- and 5`-ends. The autoradiograph is overexposed to enable visualization of very low levels of primers synthesized. Truncation of the template generally resulted in lower levels of primers; however, some changes abolished primer production. Truncation to 12 nucleotides (o12) still resulted in template activity. Truncation of the 3`-A residue of o12 to form o11B resulted in a marked reduction in the amounts of primer synthesized, although the size of the primers was unchanged. Truncation of the next 3`-residue (G) in o10B resulted in undetectable levels of primer synthesis. Deletion of CTP from the synthesis reaction had no effect using o12 as a template (data not shown); therefore, this 3`-G residue is not copied into the primer. Restoration of the 3`-AG sequence with simultaneous truncation of the template 5`-end enabled primer synthesis; however, the major products were reduced in size to nine and eight nucleotides with the o11A and o10A templates, respectively. These results suggest that the 3`-AG sequence is important to primer synthesis, warranting further investigation (see below). Additionally, oligonucleotides could be truncated to 11 nucleotides (o11B, 3`-GCCCTCCCATC-5`; the underlined residue is the first base copied into the primer or primer position 1) and still provide a template for synthesis of the 10-mer primer. Although removal of the 5`-A residue did not allow labeling of the primer by incorporation of [-P]UTP (o9), movement of the A residue farther 3` restored labeling of a seven-nucleotide product (Fig. 3 A, o9A). These results show that a DNA as small as nine nucleotides can serve as a template for the HSV primase.


Figure 3: Size and sequence requirements for HSV primase oligonucleotide templates. The results of changes in template size and sequence on primer synthesis are indicated in A and B, respectively. The DNA template is indicated above each lane (see Table I). Size markers consisted of end-labeled poly(A) partially digested with nuclease U2 ( pA-U2). The position of the predominant 10-mer oligoribonucleotide primer is indicated with an arrowhead. ``Smiling'' of the gel in B distorts the migration of the product using the 012 template (compare with A).



The results of changes in template sequence are shown in Fig. 3 B. The wild-type oligomer template used for these studies was o12 (Fig. 3 B). The importance of the 3`-AG-5` residues at the 3`-end of the template (Fig. 3 B, o12) was seen after primase activity was abolished with changes to 3`-TT-5` (Fig. 3 B, o12TT) or to 3`-GA-5` (o12AG). In addition to its ability to synthesize smaller primers (see above), the HSV primase could produce larger products when an insertion of an extra C residue (o16A) resulted in lengthening of the 10-mer primer to 11 nucleotides.

The next set of oligonucleotide templates examined the effect of insertion of a T residue (or an A residue in the T position, S-3) in different positions within the central seven positions of the 12-base template. The results above with o9A (Fig. 3 A) showed that a change at primer position 7 (C to A in the template) did not eliminate primer synthesis and, in fact, resulted in a template with relatively high activity. Oligonucleotides S-1 through S-6 represent changes in template sequence giving rise to changes in primer residues 6 through 1, respectively. The results using oligonucleotides S-1 through S-4 suggest that a change in any of the template positions directing synthesis of primer nucleotides 3-6 result in a marked decrease, but not elimination, of primer synthesis when compared with wild-type o12 (Fig. 3 B). A change in primer position 2 (S-5) virtually eliminated primer synthesis, while one at primer position 1 (S-6) only slightly decreased the abundance of primers (Fig. 3 B). These results show that the sequence at each position within this preferred template sequence in X174 DNA contribute to the magnitude of the predominant primer synthesized.

Template Requirements for DNA-dependent ATPase Activity of HSV Helicase-Primase

To investigate whether the preferred template usage by HSV helicase-primase was determined by the ability of the enzyme to bind to the DNA template, we investigated the ATPase activity of the enzyme on various oligonucleotide substrates. The results presented in indicate that ATPase activity is more dependent upon template size than on the ability to serve as a template for primer synthesis. Several oligonucleotides that did not serve as templates for detectable direct primer synthesis activity served as excellent templates for ATPase activity (). The correlation of HSV helicase-primase NTPase activity and template size parallels the findings with T4 helicase-primase (32) and suggests that primase template activity, but not ATPase template activity, is absolutely dependent upon nucleotide sequence.

Inhibition of the Coupled Primase-Polymerase Assay by Preferred-Template Oligonucleotides

The preferred oligonucleotide templates were used to examine the function of helicase-primase in the coupled primase-polymerase assay. This assay measures HSV DNA polymerase activity that is dependent upon primer synthesis on a single-stranded X174 virion DNA template. 20, 80, 150, 300, or 2000 ng of test oligonucleotide was incubated on ice with UL5/52 for 30 min, followed by the addition of other proteins and reaction components. Titration of oligonucleotides containing the preferred primer synthesis template into the assay inhibited the polymerase activity in a concentration-dependent manner. The ICresults (in nanograms of oligonucleotide) are presented in . Oligonucleotides that could not serve as primase templates did not reduce coupled primase-polymerase activity to 50% of control levels when added at concentrations as high as 2 µg. Inhibition was confirmed to be specific for the HSV helicase-primase complex using o25, o17A, o17, and o17NEG. The oligonucleotides that inhibited the coupled HSV primase-polymerase assay (o25, o17A, and o17) did not inhibit HSV DNA polymerase-UL42 or modified T7 DNA polymerase (Sequenase) activity on a singly primed X174 template or polymerase--primase activity (data not shown). Oligonucleotide o17NEG, which did not serve as a template for primase activity, did not inhibit any of the activities (data not shown). We interpret these results to indicate that primase oligonucleotide templates compete for primase activity with the X174 DNA template, while those oligonucleotides that do not direct the synthesis of a primer do not compete. The fact that these oligonucleotides are suitable substrates for ATPase activity indicates that primase activity, but not ATPase activity, is required and limiting in the coupled primase-polymerase assay.

Oligonucleotide Templates Distinguish the Effects of UL8 and ICP8 on the HSV Helicase-Primase Complex

The UL8 component of the HSV helicase-primase complex stimulates the rate of primer synthesis by a subassembly of the UL5 and UL52 components on a X174 circular DNA template (11) and on oligonucleotide templates (Figs. 1 and 2). ICP8, the HSV single-stranded DNA-binding protein, also stimulates primer synthesis on the X174 virion DNA template by helicase-primase when it contains the UL8 protein.The mechanism by which either of these proteins stimulates the primase is unknown, although neither UL8 nor ICP8 changes the preferred primase initiation site in X174 DNA.To learn more about stimulation of primase activity by the UL8 and ICP8 proteins, we tested their effects on the 50-mer oligonucleotide template. Stimulation of primer synthesis on X174 virion DNA by the ICP8 protein can be seen in Fig. 4, although a more in-depth study is presented elsewhere.The optimum ratio of ICP8 to DNA was established using the X174 template (Fig. 4). Primer synthesis by UL5/8/52 was increased by increasing amounts of ICP8 to a maximum of one molecule of ICP8/40 nucleotides of DNA. ICP8 also stimulates the ATPase activity of UL5/8/52 on X174 DNA and is optimum at a similar ratio of ICP8 to DNA.ICP8 inhibited primer synthesis at a level of one molecule/20 nucleotides, the level that is optimum for stimulation of HSV polymerase (28) . These results are consistent with the ICP8 molecule binding 12-40 nucleotides of DNA (33, 34) , thereby excluding the binding of helicase-primase.


Figure 4: HSV ICP8 single-stranded DNA-binding protein stimulates primer synthesis on X174 DNA templates in a dose-dependent manner. Primer synthesis reactions used one ICP8 molecule/20, 40, 80, 160, 320, and 640 nucleotides of X174 DNA (30 µg/ml nucleotide) to determine the optimum ratio of ICP8 to DNA in lanes 2-7, respectively. Lane 8 contained no ICP8, and lane 9 contained the UL5/52 subassembly alone. The migration of the predominant primer is indicated with an arrowhead. Migration of end-labeled poly(A) partially digested with nuclease U2 is indicated ( pA-U2).



To compare the effect of ICP8 on primer synthesis, we varied the DNA template concentration at this optimum ratio of one ICP8 molecule/50 nucleotides (Fig. 5). Primer synthesis on the X174 DNA template was increased by ICP8 at various ICP8/DNA concentrations (Fig. 5 A). In contrast, on the o50 template, the level of primers was unaffected by the addition of ICP8 (Fig. 5 B). This finding differs from that with UL8, where stimulation of primer synthesis occurs on both X174 circles and oligonucleotide templates (Fig. 2). These results suggest that the mechanism of stimulation of primer synthesis differs between the UL8 and ICP8 proteins because the stimulation by ICP8 may depend on the nature of the DNA template.


Figure 5: HSV ICP8 single-stranded DNA-binding protein stimulates primer synthesis on X174 but not oligonucleotide templates. Shown are primer synthesis reactions with ICP8 absent ( (-) ICP8) or present ( (+) ICP8) at a constant ratio of one molecule/50 nucleotides of DNA. In A, serial 2-fold dilutions of the X174 virion DNA template were made from 150 ng of nucleotide in lanes 1 and 7. In B, serial 2-fold dilutions of the o50 template were made from 7.5 pmol of molecules in lanes 1 and 6. The migration of the predominant 10-mer primer is indicated with an arrowhead.



UL8 Stimulation of Primase Activity Using Oligonucleotide Templates

The stimulation of primer synthesis by UL8 was examined further through kinetic analysis using different amounts of the oligonucleotide DNA template. Previously, a X174 DNA template was used to show that UL8 increased the rate of primer synthesis by a subassembly of the UL5 and UL52 proteins (3-fold), but did not change the Kfor DNA (11) . Using various amounts of oligonucleotide o50 as a template, kinetic analysis revealed that the rate of primer synthesis by UL5/52 ( V) on the oligonucleotide template was increased in the presence of UL8 10-fold from 0.6 to 5.9 relative absorbance units. In addition, the Kfor the DNA template was increased using the oligonucleotide from 3.3 µ M without UL8 to 8.8 µ M with UL8. This increase in Kfor DNA upon the addition of UL8 is consistent with filter binding studies using fragments of X174 DNA,suggesting that primer synthesis on oligonucleotide templates may be more sensitive to UL8-induced changes in helicase-primase than is primer synthesis on circular templates. The fact that stimulation of primer synthesis by the UL8 subunit is greater on the oligonucleotide template suggests that this template may provide a better substrate for further analysis of the mechanism of UL8 activity than circular DNA molecules.


DISCUSSION

The HSV helicase-primase complex displays relatively low specific activity in vitro on a poly(dT) DNA template (4, 7) . Additionally, the UL8 component of helicase-primase stimulates activity on natural but not homopolymer templates (9) .These results first indicated that the activity of the complex or its subunits may display template specificity. We sought to determine the template specificity for primer synthesis by helicase-primase to further examine its activity in vitro and to distinguish the effects of various stimulatory factors including the UL8 component (11) and ICP8.

We mapped a single predominant site of HSV helicase-primase primer synthesis on the X174 DNA template. This site was preferentially utilized regardless of whether the UL5/52 subassembly or the UL5/8/52 heterotrimer was used (Ref. 11 and this report). DNA oligonucleotides containing the X174 sequence at this site were used to confirm the mapping and to examine the requirements for primer synthesis. Template secondary structure was not required as oligonucleotides with virtually no potential for secondary structure could still direct specific primer initiation. Specificity was not at the level of binding to the DNA as the DNA-dependent ATPase activity of the helicase-primase complex was independent of the preferred template sequence. These results do not, however, exclude the possibility of functionally separate single-stranded DNA-binding sites for the different activities. This theory is consistent with demonstration of weak ATPase activity of the isolated UL5 subunitand localization of the primase activity within the UL52 subunit (12, 13) .

The predominant template sequence in X174 DNA was found to be 3`-AGCCCTCCCA-5`, with primer synthesis beginning at the 3`-most C residue (underlined; primer position 1). A 10-nucleotide [-P]UTP-labeled primer was produced on the X174 molecule and on oligonucleotides long enough to support its synthesis. Only the 3`-penultimate G residue of the template was absolutely required for primer synthesis; substitutions at any one of the other positions decreased but did not eliminate primer synthesis. Substitution of the template C residue at the 4th position from the 3`-end (primer position 2) virtually eliminated primer synthesis. Substitution of the C residue at primer position 1 had relatively little effect, while substitutions at any of the primer positions 3-6 decreased but did not eliminate primer synthesis. Although primer levels were increased by extension of the preferred template at either the 3`- or 5`-ends, the smallest template tested, nine nucleotides (Fig. 3 A, o9A), yielded detectable specific primer synthesis.

Although secondary structure contributes to DNA template recognition by several characterized primases, these enzymes also display specificity for nucleotide sequence of the template (reviewed in Ref. 17). These include E. coli dnaG primase recognition of the sequence 3`-GTC-5` with synthesis of a primer initiating with 5`-pppAG-3` (18, 19) , T4 primase recognition of 3`-TTG-5` or 3`-TCG-5` with synthesis of a primer initiating with 5`-pppAC-3` or 5`-pppGC-3` (35, 36) , T7 primase recognition of 3`-CTGG(T/G) or 3`-CTGTG-5` with synthesis of a primer initiating with 5`-pppACC(A/C)-3` or 5`-pppACAC (22, 37) , and DNA polymerase--primase recognition of 3`-CPyPy-5` (where Py is pyrimidine) with synthesis of a primer initiating with purine residues (23) .

HSV primase is similar to other characterized primases in that it initiates with a purine residue. However, while the prokaryotic enzymes do not tolerate changes in the consensus template sequences, changes in individual template residues, aside from the 3`-G residue, did not eliminate HSV primase activity. Another characteristic that was found to be consistent with other primases is that recognition of the template by HSV primase required a residue at the 3`-side of the primer initiation sequence that was not copied into the primer. This G residue, in the case of HSV primase, could not be truncated or substituted by an A or T residue. The fact that an oligonucleotide extending only 2 bases past this 3`-G residue was able to serve as a template distinguished HSV primase from E. coli and T7 primases, which require at least six nucleotides 3` to the 3`-GTC-5` initiation site (18, 22) .

The site of primer synthesis by HSV helicase-primase on X174 DNA mapped in this study is only the most predominant, although other sites are used (11) . We believe, however, that primer synthesis at this site accurately reflects the activity of the enzyme in vitro. Perhaps a number of factors contributed to the preference for this initiation site including sequence of flanking DNA as well as labeling conditions. As with other primases, other sites may be preferred under different reaction conditions (23, 35) . Nevertheless, sites selectively used under particular labeling conditions are a subset of those utilized under nonselective conditions where all primers synthesized can be examined (35) . In addition, the sequence mapped in this study also served as a template when labeling with [-P]CTP (Fig. 2 B). Finally, the levels of primers synthesized on the predominant template correlated with the amount of primase activity in the coupled primase-polymerase assay under nonselective conditions (11) . More detailed analyses, including those that directly examine the sequence of primers made at many template sites (37) , are needed to determine the exact template sequence requirements for HSV primase. It is important to note that concurrent with this work, Gottlieb and Challberghave determined the preferential sites of HSV primase initiation on a single-stranded E. coli vector (pBluescript) using [-P]CTP labeling. The template sequences mapped in their work have nucleotide sequences similar to that of the template mapped here.

Oligonucleotides that contained the preferred primase template sequence of HSV helicase-primase were used in further studies of the enzyme. We found that although oligonucleotides may serve as equivalent substrates for the ATPase activity (and therefore binding) of HSV helicase-primase, only those containing a specific initiation site competed for coupled primase-polymerase activity. Therefore, the primase and not the ATPase activity of the enzyme appeared to be limiting for DNA synthesis on single-stranded templates. Our results could be due to temporal sequestration of the enzyme by oligonucleotides containing the template sequence during synthesis of a primer. To determine the precise role of the ATPase, helicase, and primase functions of helicase-primase, more complex systems, such as in vitro models of HSV rolling circle replication (38) , will be required.

Other primases have shown template-specific activity and changes in specificity depending on the presence of various subunits (18, 22, 23, 36, 39, 40) . We found, however, that neither the UL8 component of helicase-primase nor the ICP8 single-stranded DNA-binding protein altered the preferential site of initiation on the X174 DNA molecule (11) .Therefore, the mechanism of in vitro stimulation of primer synthesis by these proteins is unrelated to initiation site specificity. The stimulatory effects of the two proteins, however, were distinguishable using primer synthesis on oligonucleotide templates. Kinetic studies were used to show that UL8 increased the rate of primer synthesis on oligonucleotide templates, similar to the findings using a circular template (11) . This demonstrated that the effect of UL8 was likely to be independent of template secondary structure and nucleotide length. While the ICP8 protein requires the helicase-primase complex containing UL8 for its stimulatory effect, ICP8 did not increase primer synthesis on oligonucleotide templates at a range of concentrations. These results suggest that the UL8 component and the ICP8 protein act by different mechanisms to stimulate primer synthesis. Consistent with these results is the finding that the stimulatory properties of UL8 and ICP8 differ with respect to the ATPase activity of HSV helicase-primase using various effector DNA templates.Further studies are needed to define the different mechanisms by which these proteins stimulate primer synthesis.

  
Table: Oligonucleotides used as templates


  
Table: ATPase activity and inhibition of coupled primase-polymerase activity using oligonucleotide DNAs



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: Dept. of Virology, Bristol-Myers Squibb Pharmaceutical Research Inst., 5 Research Pkwy., Wallingford, CT 06492. Tel.: 203-284-7846; Fax: 203-284-6088.

The abbreviations used are: HSV, herpes simplex virus type 1; PAGE, polyacrylamide gel electrophoresis.

J. J. Crute, personal communication.

R. K. Hamatake, M. Bifano, W. W. Hurlburt, and D. J. Tenney, manuscript in preparation.

J. Gottlieb and M. D. Challberg, personal communication.

D. J. Tenney and R. K. Hamatake, unpublished observations.


ACKNOWLEDGEMENTS

We are indebted to I. Robert Lehman for the generous gift of recombinant baculoviruses expressing HSV helicase-primase and HSV DNA polymerase proteins and to Alan Wahl for providing HeLa S3 cells and suggestions for isolation of polymerase--primase. We appreciate insightful discussions during the course of this study with Carolyn DiIanni and Richard Colonno and suggestions regarding large-scale baculovirus infections from Steven Weinheimer. We also extend thanks to Patrick McCann for sequencing ladders and to James Crute, John Gottlieb, and Mark Challberg for communication of results prior to publication.


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