From the
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
Replication of herpes simplex virus type 1 (HSV)
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 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.
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-
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.
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.).
V
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
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) .
We mapped a single predominant site of HSV helicase-primase
primer synthesis on the
The predominant template sequence in
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-
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
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
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-
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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).
(
)
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) .
(
)
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.
(
)
-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.
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.
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.
and K
values
were determined using KinetAsyst II software (IntelliKinetics, Inc.),
which fit the amount of primers synthesized ( v) to the
following equation: v =
V
S/( K
+
S). V
is expressed in absorbance units from
scanning laser densitometry of autoradiographs, and
K
for 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 MgCl
to
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.
The Preferred HSV Primase Initiation Site in
We reported previously that HSV helicase-primase
synthesizes primers at predominant sites within X174
Virion DNA
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.
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 IC
results (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 K
for 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 K
for
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 K
for 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.
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.
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 subunit
and
localization of the primase activity within the UL52 subunit
(12, 13) .
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.
-primase recognition of 3`-CPyPy-5` (where Py is
pyrimidine) with synthesis of a primer initiating with purine residues
(23) .
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 Challberg
have 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.
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
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.