|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 48, 32154-32157, November 27, 1998
From the The rate of unwinding of duplex DNA by the herpes
simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was
determined by measuring the rate of appearance of single strands from a
circular duplex DNA containing a 40-nucleotide 5' single-stranded tail, i.e. a preformed replication fork, in the presence of the
HSV-1 single strand DNA-binding protein, infected cell protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+
dependence was a reflection of both the requirement for ICP8 for
helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single
strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1
primosome was also determined indirectly by measuring the rate of
leading strand synthesis with a preformed replication fork as template
in the presence of the T7 DNA polymerase. The value of 60-65 base
pairs unwound/s by both methods is consistent with the rate of 50 base
pairs/s estimated for the rate of fork movement in vivo
during replication of pseudorabies virus, another herpesvirus.
Interaction with the helicase-primase did not increase its helicase activity.
The 152-kb1 genome of
herpes simplex virus type 1 (HSV-1) encodes three enzymes, in addition
to a single strand DNA-binding protein, that are required for its
replication (1, 2). These include a heterodimeric, highly processive
DNA polymerase (3-6), an origin-binding protein with 3'-5'-helicase
activity (7-9), and a heterotrimeric primosome with both helicase and
primase activities (10-13). Earlier studies of the helicase activity
of the helicase-primase showed its rate of unwinding of duplex DNA substrates to be 2 bp/s (12), a rate far below the 50 bp/s estimated for the rate of replication of pseudorabies virus, a related
herpesvirus (14).
The HSV-1 helicase-primase very likely exists in association with
other HSV-1-encoded enzymes as part of a replisome (15, 16). Several
structural and functional interactions between the components of the
putative replisome have been described (1). For example, the single
strand DNA-binding protein, infected cell protein 8 (ICP8) interacts
with the UL8 subunit of the helicase-primase (17). It was recently
demonstrated that the catalytic subunit of the HSV-1 DNA polymerase
also interacts with the UL8 subunit (18). In light of these
observations, we undertook an examination of the effect of the
HSV-1-encoded DNA polymerase on the helicase rate. Studies of the
Escherichia coli DnaB helicase have shown its rate to be
strongly influenced by its association with its cognate DNA polymerase
III holoenzyme (19). We show here that the helicase activity of the
HSV-1 helicase-primase in the presence of the HSV-1 single strand
DNA-binding protein ICP8 is extraordinarily sensitive to reaction
conditions, and at the appropriate Mg2+ conditions and
ionic strength, the rate approaches the rate of replication fork
movement in vivo. However, this rate is not significantly influenced by its association with the HSV-1 DNA polymerase.
Enzymes--
The helicase-primase, DNA polymerase-UL42 protein,
and ICP8 were purified by previously described procedures (17).
DNA Helicase Substrates--
The formation of the 20-bp helicase
substrate (Fig. 1, Structure A) has been described
previously (17). The circular duplex DNA substrate containing a
preformed replication fork was generated by annealing a 60-base
oligonucleotide (15 pmol,
5'-ACATGATAAGATACATGGATGAGTTTGGACAAACCACAACGTAAAACGACGGCCAGTGCC-3') to 5 pmol of M13mp18 single-stranded DNA (Biolabs) to generate a
20-bp double-stranded region with a 40-nucleotide unpaired 5' tail. The
single-stranded circle was converted to the duplex form (Fig. 1,
Structure B) by incubating the tailed M13mp18 single stranded DNA (10 pmol) with ICP8 (1 nmol) and HSV-1 DNA polymerase (100 pmol) in a reaction mixture (500 µl) containing 20 mM
Tris-HCl, pH 7.3, 10% glycerol, 4 mM dithiothreitol, 0.5 mM ATP, 200 µg/ml bovine serum albumin, 4.5 mM MgCl2, 0.1 mM dATP, dCTP, dGTP,
and dTTP, and 50 mM NaCl. Incubation was at 37 °C for 90 min, and the reaction was stopped by the addition of 125 µl of 2%
SDS and 0.8 mg/ml proteinase K, followed by further incubation at
37 °C for 30 min.
Linear double stranded DNA was generated by cleaving plasmid pTZ18r
(Amersham Pharmacia Biotech) with the EcoRI and
SmaI restriction enzymes to yield a linear double-stranded
DNA, 2860 bp in length, containing a 4-base overhang at the 5' end and
a 3' blunt end. Both the circular duplex DNA with a preformed
replication fork and the linear pTZ18r DNA were extracted first with
phenol/chloroform and then with chloroform and precipitated by the
addition of ammonium acetate to 2.5 M and 2.5 volumes of
ethanol. The DNAs were kept at 4 °C for 30 min, centrifuged, washed
with ice-cold 70% ethanol, dried, and dissolved in 10 mM
Tris-HCl, pH 8.0, 0.1 mM EDTA.
Assay of DNA Helicase Activity--
The reaction mixture (20 µl) contained either 0.1 µg (21 fmol) of circular duplex M13mp18
DNA with a preformed replication fork (Fig. 1, Structure B)
or 0.1 µg (48 fmol) of linear pTZ18r, 20 mM Tris-HCl, pH
7.6, 10% glycerol, 3 mM dithiothreitol, 3 mM ATP, 100 µg/ml bovine serum albumin, and the indicated concentration of MgCl2. Twenty pmol of ICP8 and 1 pmol of
helicase-primase were added. Incubation was at 34 °C for the times
indicated and stopped by addition of 4 µl of stop solution (90 mM EDTA, pH 8.0, 6% SDS, 30% glycerol, 0.25% bromphenol
blue, 0.25% xylene cyanol). The products were separated by
electrophoresis at 150 V for 3 h through an 0.8% agarose gel with
Tris borate/EDTA (89 mM Tris borate, 1 mM EDTA)
containing 1.0 µg/ml ethidium bromide. The DNA was denatured by
submerging the gel in 900 ml of denaturation buffer (1.5 M
NaCl, 0.5 M NaOH) for 30 min with gentle agitation. The gel
was rinsed with water for 5 min and then neutralized by gentle agitation in 900 ml of neutralization buffer (1.5 M NaCl, 1 M Tris-HCl, pH 7.4) for 30 min. The DNA was transferred by
a Hybond-N nylon membrane (Amersham Pharmacia Biotech) using 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.5)
overnight. It was covalently bound to the filter by UV irradiation and
then preincubated for 60 min at 65 °C with 50 ml of prehybridizing
buffer (5 × SSC, 0.5% SDS, 0.1 g of bovine serum albumin,
0.1 g of Ficoll 400, 0.1 g of polyvinylpyrrolidone) before
adding a randomly primed probe (Stratagene Prime-it II) for M13mp18 or
pTZ18r. After incubation overnight, the filter was washed for 30 min
with 0.1% SDS and l × SSC at 65 °C. The labeled hybrids were
visualized by autoradiography. Measurement of helicase activity with
Substrate A (Fig. 1) was performed as described previously (17).
Measurement of Leading Strand Synthesis--
The reaction
mixture (50 µl) contained 30 mM Hepes-NaOH, pH 7.6, 3 mM dithiothreitol, 8 mM magnesium acetate, 4 mM ATP, 100 µM dATP, dGTP, and dTTP, 10 µM dCTP, 5 µCi of [ Measurement of DNA Polymerase Activity--
The reaction mixture
(50 µl) contained 30 mM Hepes-NaOH, pH 7.6, 3 mM dithiothreitol, 8 mM magnesium acetate, 4 mM ATP, 100 µM dATP, dGTP, and dTTP, 10 µM dCTP, 5 µCi of [ Effect of Mg2+ Concentration on the Helicase Activity
of the HSV-1 Helicase-primase--
To measure the helicase activity of
the HSV-1 helicase-primase, a circular duplex DNA with a preformed
replication fork was used (Fig. 1,
Structure B). Our earlier studies had shown that ICP8 is
essential for the unwinding of long stretches of duplex DNA by the
helicase-primase, presumably as a consequence of its ability, as a
helix-destabilizing protein, to prevent reannealing of the separated
single strands (17). Saturating amounts of ICP8 (one ICP8 monomer/12
nucleotides of single-stranded DNA) were therefore included in all
reactions with this substrate. Examination of the helicase activity at
various ionic strengths showed 20 mM NaCl to be optimal
(data not shown). However, as shown in Fig.
2A, in the presence of 3 mM ATP unwinding was observed only at a narrow range of
Mg2+ concentrations, between 1.0 and 3.5 mM.
The narrow range of Mg2+ concentrations at which helicase
activity could be observed prompted us to examine the effect of
Mg2+ concentration on the other component of the reaction,
ICP8. ICP8 can promote the annealing of homologous single strands of
DNA (20, 21). Earlier studies had in fact shown this reaction to be
sensitive to Mg2+ concentration (21). As substrate, the
circular duplex DNA with a preformed replication fork, which had been
heated to 100 °C for 2 min and then quickly chilled on ice, was
used. The experimental conditions, including 3 mM ATP, were
identical to those described for the previous experiment. As shown in
Fig. 2B, the single strands reannealed in the presence of
ICP8 at Mg2+ concentrations of The HSV-1 Helicase-primase Can Unwind Duplex DNA at the Rate of 60 bp/s--
To estimate the rate of DNA unwinding catalyzed by the HSV-1
helicase-primase, we performed the reaction under conditions that were
found to be optimal for helicase activity (20 mM NaCl, 3 mM Mg2+, 3 mM ATP, saturating
ICP8). The molar ratio of enzyme to substrate was 40:1 for the circular
substrate and 20:1 for the linear substrate. The complete unwinding of
the circular duplex with a preformed replication fork (7240 bp)
occurred in 2 min (Fig. 4A).
The rate of unwinding was therefore 60 bp/s. This value is sufficient
to support the rate of DNA replication observed in vivo with
pseudorabies virus.
With a linear duplex substrate (linear pTZ18r DNA), the rate was 24 bp/s (Fig. 4B). The lower rate is presumably attributable to
the lack of a sufficiently long single-stranded loading site for the
helicase-primase.
As an alternative approach to measurement of the rate of unwinding of
duplex DNA by the helicase-primase, the rate of leading strand DNA
synthesis by the T7 DNA polymerase coupled to the helicase action of
the helicase-primase in the absence of ICP8 was determined. This method
depends on the inability of the T7 DNA polymerase to replicate through
the regions of duplex DNA (22, 23) and has the advantage of being
independent of the reannealing reaction, because DNA synthesis and
unwinding are closely coordinated.
Because a functional interaction between the HSV-1 helicase-primase and
the T7 DNA polymerase is unlikely, the rate of the T7 DNA
polymerase-catalyzed leading strand synthesis should reflect the rate
of unwinding of the DNA duplex by the helicase-primase. We found that
at 4 min the 7-kb substrate had been extended by the T7 DNA polymerase
to yield products of ~23 kb. The rate of unwinding was calculated to
be 65 bp/s, in excellent agreement with the direct measurements of
helicase activity (60 bp/s) (Fig. 5).
The HSV-1 DNA Polymerase Does Not Stimulate the Helicase Activity
of the Helicase-primase--
Studies of the dnaB helicase of E. coli have shown it to be markedly stimulated by its interaction
with the DNA polymerase III holoenzyme (19). We therefore wished to
determine whether there was a comparable stimulation of the
helicase-primase by the HSV-1 DNA polymerase. In a reaction with
helicase-primase, the DNA polymerase-UL42 protein, and ICP8, leading
strand synthesis was observed (Fig. 5). Under these conditions we found
no evidence for lagging strand synthesis. With the HSV-1 DNA
polymerase-UL42 protein, 12 min were required to extend the 7-kb
substrate to a length of ~23 kb. The rate was therefore 20 bp/s. This
rate was similar to the rate of DNA synthesis by the DNA
polymerase-UL42 protein in the presence of ICP8 with a primed M13mp18
single stranded DNA template (Figs. 1, Structure A, and
6). In this instance the synthesis of the
7.2-kb duplex DNA was completed within 10 min. Thus, leading strand
synthesis by the DNA polymerase does not enhance the rate of unwinding
of a DNA duplex by the helicase-primase. Moreover, deoxynucleotide
polymerization by the HSV-DNA polymerase appears to be rate-limiting
during leading strand synthesis.
Thus far we have not found evidence for a coupled leading and lagging
strand synthesis in these reactions. Possibly additional factors are
required to promote synthesis of the lagging strand.
*
This work was supported in part by National Institutes of
Health Grant AI-26538 (to I.R.L.) and Swedish Cancer Society Grant 2552-B97-11XAC (to P.E.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by a grant from the Swedish Institute.
The abbreviations used are:
kb, kilobase(s); bp, base pair(s); HSV-1, herpes simplex virus type 1; ICP8, infected cell
protein 8.
The Herpes Simplex Virus Type 1 Helicase-primase
ANALYSIS OF HELICASE ACTIVITY*
§¶,
Department of Biochemistry, Beckman Center,
Stanford University, Stanford, California 94305-5307 and
§ Department of Medical Biochemistry, Goteborg University,
S-413 90 Goteborg, Sweden
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-32P]dCTP (3000 Ci/mmol), 250 µM CTP, 250 µM GTP, 250 µM UTP, 40 mM creatine phosphate, 5 µg of
creatine kinase, and 40 fmol of M13mp18 circular duplex DNA with a
preformed replication fork (Fig. 1, Structure B). Where
indicated, 1 pmol of helicase-primase, 10 pmol of ICP8, 500 fmol of
HSV-1 DNA polymerase-UL42 protein, or 0.01 unit of T7 DNA polymerase
was added. Incubation was at 37 °C for the times indicated and
stopped by the addition of 5 µl of 8% SDS and 2.4 µg/ml proteinase
K, followed by incubation for 30 min at 37 °C. Twelve µl of
denaturing gel loading buffer (50 mM NaOH, 1 mM
EDTA, 0.25% bromphenol blue, 0.25% xylene cyanol) and 1 µl of 0.5 M EDTA were added before loading half the reaction mixture
onto a 0.7% denaturing agarose gel (50 mM NaOH, 1 mM EDTA). The gels were run at 1 V/cm for 20 h, dried,
and autoradiographed with an intensifying screen.
-32P]dCTP (3000 Ci/mmol), and 20 fmol of tailed M13 mp18 single-stranded DNA (Fig. 1,
Structure A). Ten pmol of ICP8 and 500 fmol of HSV-1 DNA
polymerase-UL42 protein were added. Incubation was at 37 °C. Twelve
µl of stop solution (90 mM EDTA, pH 8.0, 6% SDS, 30%
glycerol, 0.25% bromphenol blue, 0.25% xylene cyanol) were then added
to 25 µl of the reaction mixture, and the samples were analyzed gel electrophoresis in 0.8% agarose gels containing Tris borate/EDTA with
1.0 µg/ml ethidium bromide. The gels were run at 150 V for 3 h.
The dried gels were autoradiographed with an intensifying screen.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
View larger version (13K):
[in a new window]
Fig. 1.
Helicase substrates. The circular duplex
DNA with a preformed replication fork was generated by annealing a
60-base oligonucleotide to single-stranded M13 mpl8 DNA (Structure
A). The 20-bp duplex region was then extended by reaction
with HSV-1 DNA polymerase-UL42 protein and ICP8 (Structure
B). See "Materials and Methods" for details.
View larger version (90K):
[in a new window]
Fig. 2.
Effect of Mg2+ concentration on
the helicase activity of the helicase-primase in the presence of
ICP8. Reaction mixtures were prepared as described under
"Materials and Methods," except that increasing amounts of
MgCl2+ were added as indicated. A, helicase
activity of the helicase-primase on the circular duplex DNA with a
preformed replication fork (Fig. 1, Structure B). Twenty
pmol of ICP8 and 1 pmol of helicase-primase were added to each
reaction. First lane, substrate heated to 100 °C and then
chilled. B, reannealing of separated single strands.
Structure B was heated to 100 °C and then rapidly chilled. Twenty
pmol of ICP8 were added to each reaction mixture. The products were
subjected to 0.8% agarose gel electrophoresis, transferred to a nylon
filter, and hybridized with a random probe as described under
"Materials and Methods." First lane, untreated
substrate. S, substrate; P, product.
3.5 mM. At
Mg2+ concentration <3.5 mM, no ICP8-mediated
reannealing was observed. The reaction was dependent on ICP8, because
no reannealing of the single strands was observed in an identical
experiment performed in the absence of ICP8 (results not shown). The
range of Mg2+ concentrations at which ICP8 could promote
the reannealing of single strands coincided with the concentrations at
which no helicase activity could be detected. It therefore appears that
the apparent inhibition of helicase activity at Mg2+
concentrations >3.5 mM is simply a consequence of the
reannealing of the product single strands under these conditions. In
contrast, with the 20-bp helicase substrate (Fig. 1, Structure
A) for which ICP8 is not required (17), helicase activity
was observed at all Mg2+ concentrations tested (Fig.
3).
View larger version (57K):
[in a new window]
Fig. 3.
The helicase activity of helicase- primase in
the absence of ICP8 is independent of Mg2+
concentration. Reaction mixtures were prepared as described under
"Materials and Methods," except that 500 fmol of helicase-primase
were added and increasing amounts of MgCl2 were added as
indicated. The products were subjected to 12% polyacrylamide gel
electrophoresis as described under "Materials and Methods."
S, substrate; P, product. First lane,
untreated substrate; second lane, substrate heated to
100 °C and then chilled.
View larger version (81K):
[in a new window]
Fig. 4.
Measurement of helicase rate of the HSV-1
helicase-primase. Reaction mixtures were prepared as described
under "Materials and Methods." Unless otherwise indicated, all
reactions contained 1 pmol of helicase-primase and 20 pmol of ICP8.
A, helicase assay using circular duplex DNA with a preformed
replication fork (Fig. 1, Structure B) as substrate.
B, helicase assay using linear double-stranded DNA (pTz18r)
as substrate. The products were subjected to 0,8% agarose gel
electrophoresis, transferred to a nylon filter, and hybridized with a
random probe as described under "Materials and Methods."
First lane, untreated substrate; second lane,
substrate heated to 100 °C and then chilled; third lane,
helicase-primase incubated for 20 min without ICP8; fourth
lane, ICP8 incubated for 20 min without helicase-primase.
S, substrate; P, product.
View larger version (68K):
[in a new window]
Fig. 5.
Leading strand DNA synthesis by T7 DNA
polymerase and HSV-1 DNA polymerase-UL42 protein in the presence of
helicase-primase. Reaction mixtures were prepared as described
under "Materials and Methods." Samples (50 µl) were removed at
the times indicated and analyzed by 0.7% denaturing agarose gel
electrophoresis as described under "Materials and Methods." Size
markers were nonradioactive phage 
DNA cleaved with the
HindIII or KpnI restriction enzymes.
nt, nucleotides.
View larger version (60K):
[in a new window]
Fig. 6.
Rate of the HSV-1 DNA polymerase. The
reaction mixture was prepared as described under "Materials and
Methods." Samples (50 µl) were removed at the times indicated and
analyzed by 0.8% denaturing agarose gel electrophoresis as described
under "Materials and Methods." ssDNA, position of
single-stranded circular M13mp18 DNA; dsDNA, position of
fully double-stranded circular M13mp18 DNA.
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Biochemistry, Beckman Center, B-400, Stanford University, Stanford, CA 94305-5307. Tel.: 650-723-6161; Fax: 650-723-6783.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Mapelli, S. Panjikar, and P. A. Tucker The Crystal Structure of the Herpes Simplex Virus 1 ssDNA-binding Protein Suggests the Structural Basis for Flexible, Cooperative Single-stranded DNA Binding J. Biol. Chem., January 28, 2005; 280(4): 2990 - 2997. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Nimonkar and P. E. Boehmer Role of Protein-Protein Interactions during Herpes Simplex Virus Type 1 Recombination-dependent Replication J. Biol. Chem., May 21, 2004; 279(21): 21957 - 21965. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Nimonkar and P. E. Boehmer Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1 PNAS, September 2, 2003; 100(18): 10201 - 10206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ward, M. Falkenberg, P. Elias, M. Weitzman, and R. M. Linden Rep-Dependent Initiation of Adeno-Associated Virus Type 2 DNA Replication by a Herpes Simplex Virus Type 1 Replication Complex in a Reconstituted System J. Virol., November 1, 2001; 75(21): 10250 - 10258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mapelli, M. Mühleisen, G. Persico, H. van der Zandt, and P. A. Tucker The 60-Residue C-Terminal Region of the Single-Stranded DNA Binding Protein of Herpes Simplex Virus Type 1 Is Required for Cooperative DNA Binding J. Virol., October 1, 2000; 74(19): 8812 - 8822. [Abstract] [Full Text]
|
|
|
|
|
|
M. Falkenberg, I. R. Lehman, and P. Elias Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome PNAS, April 11, 2000; 97(8): 3896 - 3900. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Lehman and P. E. Boehmer Replication of Herpes Simplex Virus DNA J. Biol. Chem., October 1, 1999; 274(40): 28059 - 28062. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |