THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
Vol. 262, No. 9, Issue of March 25, pp. 4252-4259,1987
Printed in U.S.A.
0 1987 by The American Society of Biological Chemists, Inc.
Processive Replication of Single-stranded DNA Templates by the
Herpes Simplex Virus-induced DNA Polymerase*
(Received for publication, September 22, 1986)
Michael E.O’DonnellS, Per Eliass, and I. R. Lehman
From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305
The DNA polymerase encoded by herpes simplex
virus 1 consists of a single polypeptide of M, 136,000
that has both DNA polymerase and 3’+5’ exonuclease
activities; it lacks a 5‘+3‘ exonuclease. The herpes
polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular singlestranded DNA (turnover number -0.25 nucleotide).
Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (& e 1
nM). The single-stranded DNA-binding protein from
Escherichia coli greatly stimulates the rate (turnover
number -4.5 nucleotides) by facilitating the efficient
binding to and extension of the DNA primers. Synchronous replication by the polymerase of primed single-stranded DNA circles coated withthesinglestranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase
template without dissociation, despite the 20-30 min required to replicate
the circle. Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded
DNA circles. ATP (or dATP) is not required to initiate
or sustain highly processive synthesis. The 3‘+5‘ exonuclease associated with the herpesDNA polymerase
binds a 3’ terminus tightly ( K , < 50 nM) and is as
sensitive as the polymerase activity to inhibition by
phosphonoacetic acid (Ki -4 h ~ )suggesting
,
closecommunication between the polymerase and exonuclease
sites.
a single-stranded DNA-binding protein (2). Partial purification of the herpes DNA polymerase has shown it to be a n
approximately150-kDa polypeptide (3) in good agreement
withthe molecularmass of 136 kDapredicted from the
nucleotide sequence of the gene (4, 5). As a first step in our
analysis of HSV-1 DNAreplication, we havepurified the
herpes-induced DNA polymerase to homogeneity and examined the dynamicsof its replication of ssDNA templates.
A second viral encoded protein known to be essential for
HSV-1 DNA replication, ICP8 (6-8), binds ssDNA tightly
and cooperatively (9) and is therefore analogous to the prokaryotic single-stranded DNA-binding proteins typified by
Escherichia coli SSB and T 4 gene 32 protein (1).The interaction of ICP8 with the DNA polymerase in the presence of
single- and double-strandedDNA templates is the subject of
the accompanying paper (10).
EXPERIMENTALPROCEDURES
Materials-Unlabeled and labeled nucleotides were purchased from
PharmaciaP-L Biochemicals and Amersham Corp., respectively.
dAMP-PNP was a gift from Dr. B. Alberts (University of California,
San Francisco). $X and Ml3Goril viral DNAs were prepared as
described (11); all viral DNA concentrations are expressed as DNA
molecules and were calculated using an AZWof 1 as equivalent to 36
pg/ml. (dA)lmand (dT)17 were purchased from Pharmacia P-L
Biochemicals. Calf thymus DNA, purchased from Sigma, was activated as described (12). SSB (4 X lo4 units/mg) (13) was a gift from
Dr. D. Soltis (this department). DNA polymerase 111 holoenzyme
fraction V (7 X lo6 units/mg) was prepared as described (14). E. coli
DNA ligase was prepared as described (15). T4 DNA polymerase and
The replication of a duplex DNA chromosomerequires the T 4 polynucleotide kinase were obtained from the United States
Biochemical Co. Phosphonoacetic acid was obtained from Sigma.
concerted actionof several proteins that are thought to assemBio-Gel A-1.5m and proteinmolecular weight markers were obtained
ble into a multiprotein complex (1).T o understand the mo- from Bio-Rad.Plastic-backed
polyethyleneimine-cellulose sheets
lecular mechanism bywhich a eukaryotic chromosome is (Polygram MN300) were obtained from BrinkmannInstruments;
replicated, we have chosen to study herpes simplex virus 1 Centricon 10 was from Amicon.
Bufiers-Buffer A was 20 mM Tris-C1 (pH 7.5), 6 mM MgClz, 4%
(HSV-l).’ The HSV-1 genome which is a linear duplex apmM
proximately 150 kb in length encodes many of the enzymes glycerol, 0.1 mM EDTA, 40 pg/ml bovine serumalbumin,5
required for its replication, including a DNA polymerase and dithiothreitol. Buffer B was 20 mM Hepes/Na+ (pH 7.5), 0.5 mM
dithiothreitol, 0.5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
* This research was supported by Grant GM06196 from the Na- 10% (w/v) glycerol. Buffer C was 50 mM Tris-C1 (pH 7.5), 150 mM
tional Institutes of Health. The costs of publication of this article (NH,),SO,, 0.5 mM dithiothreitol, 0.1 mM EDTA.
Cells and Viruses”RA305 (16), a thymidine kinase-deficient muwere defrayed in part by the payment of page charges. This article
must therefore be hereby marked “advertisement” in accordance with tant of HSV-1 [F], was used to infect roller-bottle cultures of Vero
cells using a multiplicity of infection of 5-10 plaque-forming units/
18 U.S.C. Section 1734 solely to indicate this fact.
$ Fellow of the Helen Hay Whitney Foundation. Present address: cell.
Purification of HSV-1 DNA Polymeruse-The herpes-induced
Dept. of Microbiology, Cornel1 University Medical School, 1300 York
DNA polymerase was purified by a modification of the method of
Ave., New York, NY 10021.
5 Supported by a fellowship from the Knut and Alice Wallenberg Powell and Purifoy (3). The steps in thepurification up to chromatography onphosphocellulose have been described (17). Briefly, nuclei
Foundation, Sweden.
The abbreviations used are: HSV, herpes simplex virus; Hepes, were prepared from 50 roller bottles of infected cells (32 g, wet weight)
4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid; dNTPs, deoxy- and extracted with 1.7 M NaC1. The DNA was removed by ultracenribonucleoside triphosphates; MMP-PNP, 2’-deoxy-5’-adenyl imi- trifugation at 100,000 X g, and the supernatant(200 ml) was dialyzed
dodiphosphate; $X, bacteriophage 4x174; ssDNA, single-stranded for 6 h against two changes (2 liters each) of buffer B and loaded
DNA; RFI, closed circular duplex DNA; RF 11, circular duplex DNA onto a phosphocellulose column (16-ml bed volume) equilibrated with
with a nick in one strand; kb, kilobases; SDS, sodium dodecyl sulfate; buffer B. The phosphocellulose column was eluted with alinear
SSB, E. coli single-stranded DNA-binding protein; ICP8, infected
cell gradient from 0.1 to 0.6 M NaCl in a total volume of 200 ml of buffer
B. Herpes DNA polymerase activity eluted at approximately 0.3 M
protein 8.
’
4252
4253
HSV-1 DNA Polymerase
TABLE
I
Purification of HSV-1 DNA polymerase from HSV-1-infected Vero
rdls
Fraction
Total
Total
protein activitf
units x
mg
10"
Specific
activity
unitslmg
x 10-6
Overall Purifiyield cation
7%
-fold
Ia. Cytoplasm 1.9
267
0.79
12
Ib. Nuclear extract
Before dialysis
123
13.0
10.5
82
After dialysis
60
14.0
23
88
6b
4.8
61
30
16
11. Phosphocellulose
7.9
23
326
111. DNA-cellulose
0.30
3.7
1230
0.12
2.6
2200
16
583
IV. Glycerol gradient
One unit is eaual to 1 pmol of DNA synthesis in 10 min at 37 "C.
Relative to the combined fractions Ia and Ib(before dialysis).
NaC1. Polymerase fractions were pooled, dialyzed, and applied to a
5-ml ssDNA-cellulose column (prepared as described in Ref. 18)
equilibrated withbuffer B containing 30% (w/v) glycerol. The column
was eluted with a linear gradient from 0.1 to 1.0 M NaCl in a total
volume of 100 ml of buffer B containing 30% (w/v) glycerol. DNA
polymerase eluted at approximately 0.3 M NaCl. Active fractions were
pooled, dialyzed against buffer C containing 5% (w/v) glycerol, and
concentrated by ultrafiltration in a centrifuge (Centricon 10) to 0.75
mg/ml in a total volume of 400 pl.The concentratedDNA polymerase
fraction was divided into two equal portions, and each was loaded
onto a 10.6-ml linear 10-30% gradient of glycerol in buffer C. The
glycerol gradients were centrifuged a t 40,000 rpm in aBeckman
SW41.Ti rotor for 40 h at 4 "C. After centrifugation, 0.2-ml fractions
were collected from the bottom of the tubes. The purificationis
summarized in Table I. Fractions at each step of purification were
also analyzed by electrophoresis in a 7.5% SDS-polyacrylamide gel,
and proteins were visualized by staining with Coomassie Blue (19).
Protein concentration was measured by a modification of the method
of Bradford (see Ref. 20) using bovine serum albumin as a reference.
The concentration of active DNA polymerase molecules was determined from the amount of SSB-coated singly primed circular @X
ssDNA that was replicated by a given amount of polymerase in 40
min at 30 "C (assuming one polymerase molecule/primer terminus).
The number of circles replicated was calculated from the total amount
of DNA synthesis, the specific activity of nucleotides, and the size of
the primed ssDNA circle (21) (i.e. moles of nucleotide polymerized
per 5386).
Templates-The sequence and synthesis of the deoxyoligonucleotide 15-mer primers are described in a previous report (22). In most
of these studies, the @X ssDNA was primed with primer 1; in one
experiment, the ssDNA was primed with primer 4 (see text). The
M13Goril ssDNA was primed using primer 5. Hybridization of synthetic primers (2.1 p~ 15-mer) to viral ssDNA (160 nM circles) was
in 10 mM Tris-C1, 0.3 M NaCl, and 0.03 M sodium citrate (final pH
8.5). The hydridization mixture was heated briefly to 100 "C, allowed
to cool to room temperature over 20 min, and incubated a further 1
h at 30 "C.
Primed @X ssDNA labeled at the 5' terminus of the synthetic
DNA primer was made by labeling the primer with T4 polynucleotide
kinase and [ T - ~ ~ P I A Tand
P , then hybridizing the labeled primer to
@XssDNA, followed by filtration on Bio-Gel A-1.5m in 10 mM TrisC1 (pH 7.5), 0.5 mM EDTA, 100 mM NaCl to remove excess labeled
ATP and primer.
(dA)1000.(dT)17
was made by incubating (dA)lwo (0.5 mg/ml) and
(dT),, (5 pg/ml) in 10 mM Tris-HC1, 0.3 M NaC1, and 0.03 M sodium
citrate (final pH 8.5) at 30 "C for 1 h.
Synthetic 3'end-labeled hairpin templates (synthetic 57-mers)
with either a paired 3'-deoxyadenylate or unpaired 3"deoxythyrnidylate were generously provided by Hisaji Maki (thisdepartment).'
Measurement of DNA Synthesis-For the assay of DNA polymerase activity during purification, reaction mixtures (25 pl) contained
2.2 pg of activated DNA, 0.5 mM ATP, 60 p~ each dATP, dCTP, and
dGTP, 5 p M [3H]dTTP (40 Ci/mmol), and 150 mM (NH,),SO, in
buffer A contalnlng 3 mM MgCl,. The reaction was initiated by
adding 1 pl of enzyme fraction containing up to 30 units of DNA
polymerase. Incubation was for 10 min at 37 "C. Reactions were
quenched with 1 ml of cold 10% trichloroacetic acid, 0.1 M pyrophos-
H. Maki and A. Kornberg, manuscript in preparation.
phate. Incorporation of labeled nucleotide into acid-insoluble material
was measured as described (22).
Specific details of DNA synthesis on singly primed @XssDNA are
given in the figure legends. Reactions were incubated at 30 "C, and
samples taken at the timesindicated were quenched by adding them
to an equal volume of 1%SDS, 40 mM EDTA. The samples, after
quenching, were usually divided into two parts. One-half was analyzed
for DNA synthesis by measuring the total nucleotide incorporated
into acid-insoluble material as described (22). (The values for DNA
synthesis refer to the entire reaction mixture). The other half was
used for the analysis of the replication products by electrophoresis in
neutral 0.8% agarose gels as described (22).Forautoradiography,
dried gels were exposed to Kodak XAR-5 x-ray film.
Measurement of3"Hj' Exonuclease Actiuity-Synthetic DNA hairpin templates containing either paired or unpaired 3"labeled termini
were used as substratesfor measurement of 3'+5' exonuclease activity. Reaction mixtures (12.5 pl) contained 3.1 pmol of hairpin template, 83 fmol of DNA polymerase, and 60 mM NaCl in buffer A.
When present, dNTPs were 60 p~ each. Incubation was at 30 "C.
Samples (1 pl) were quenched instantly upon being spotted onto
polyethyleneimine-cellulose strips containing ATP and ADP as carrier and developed in 1 M formic acid and 0.5 M LiCl. Positions of
unused substrate (origin) and dTMP or dAMP (near the solvent
front) were identified by autoradiography andcut out, andtheir
radioactivity was determined by scintillation counting.
RESULTS
HSV-1 DNA Polymerase Is Stimulated by E. coli SSBThe HSV-1 DNA polymerase was only minimally active with
circular 4X ssDNA (5.4 kb) primed with a synthetic 15-mer
(Fig. 1).Activity was, however, stimulated more than 20-fold
upon coating the primed ssDNA with E. coli SSB (Fig. 1).
The herpesDNA polymerase was also stimulated (1.5-5-fold)
by 250 mM NaCl (data not shown), the extentof stimulation
depending upon the
relative amounts of DNA polymerase and
DNA in the assay.
The influence of ionic strength onpolymerase activity when
the herpes DNA polymerase was in molar excess over singly
primed ssDNA circles is shown in Fig. 2. DNA polymerase
activity was stimulated %fold by 150 mM NaCl at early times
10
20
30
40
50
60
MINUTES
FIG. 1. Stimulation of HSV-1 DNA polymerase by E. coli
SSB. Herpes DNA polymerase (13 fmol) was incubated with 78 ng
of singly primed 4X ssDNA (45 fmol as circles) in buffer A (168 pl)
containing 60 p~ each dCTP and dGTP. The reaction mixture was
incubated 3 min and then replication was initiated by addition of 7
plof1.5
mM dATP and 0.5 mM [a-32P]d'M'P (2400 cpm/pmol).
Samples (20 pl) were removed a t the times indicated, quenched, and
analyzed for DNA synthesis as described under "Experimental Procedures." A, no additions; 0, ssDNA was coated with 0.78 pg of SSB.
HSV-1 DNA Polymerase
4254
of SSB, theherpes DNA polymerase was maximally active on
SSB-coated ssDNA at anionic strength of approximately 70
mM (data notshown).
HSV-1 DNA Polymerase Is Highly Processiue-DNA synthesis by the herpes DNA polymerase withSSB-coated
25
ssDNA as template reached a plateau value after 20-30 min
even though all of the available DNA had notbeen replicated
-(Fig. 1). Moreover, the extent of DNA synthesis was proportional to the amount of DNA polymerase added (data not
- 20
II
shown). The limited DNA synthesis suggests a highly procesY
sive mode of nucleotide polymerization wherein each polymX
erase molecule completely extends a DNA primer around the
5> 15
ssDNA circle without dissociation and is slow in cycling to
a
another
primed template.
z
n
Analysis
of replication products from singly primed SSB10
coated ssDNA by native agarose gel electrophoresis supports
the highly processive mode of nucleotide polymerization (Fig.
3B). During the time in which full-length products (RF 11)
5
were formed, most of the primed ssDNA remained unchanged
(detected by UV-induced ethidium bromide fluorescence).
The 20 min required for the complete replication of a 4X
ssDNA molecule (5.4 kb) yields an average turnover number
15
10
5
of 4.5 nucleotides/s/polymerase molecule. The lack of signifMINUTES
icant radioactivity in theregion of the gel between the primed
FIG. 2. Influence of ionic strength on replication of singly ssDNA and RFI1 product after 30 min indicates that cycling
primed &X ssDNA. Reaction mixtures (125 pl) contained 204 fmol
of singly primed @XssDNA (as circles), 500 fmol of herpes DNA of the polymerase to otherprimed ssDNA molecules is slow.
polymerase, 0.5 mM ATP, 60 p~ dCTP, dGTP, and dATP, and 20 That the remaining primed circles were effective templates
was demonstrated by their replication upon further addition
p~ [m3*P]dTTP(3000 cpm/pmol) in buffer A. Samples (20 pl) were
removed at the indicated times, quenched, and analyzed for DNA of DNA polymerase (not shown).
synthesis as described under “Experimental Procedures.” The conThe herpes DNA polymerase was also highly processive in
centration of NaCl in individual reactions was: A, 0; 0 , 3 0 mM; A, 60 the absence of SSB. An agarose gel analysis of the replication
mM; 0,90 mM; 0, 120 mM; 0 , 1 5 0 mM.
products formed with singly primed 4X ssDNA showed that
A ) NO ADDITION
B) SSB ADDED
most of the DNA templates had not reacted; nevertheless,
discrete, partially replicated species and some fully replicated
MIN: 10 2 0 40 60
MIN: 3
6 10 2 0 30
RF I1 molecules were evident (Fig. 3A).
The processivity of the HSV-1 DNA polymerase was demcRFll
a-RFII
onstrated in a second type of experiment diagramed in Fig.
4A. The DNA polymerase was preincubated with an 18-fold
excess of singly primed +X ssDNA coated with SSB so that
each polymerase molecule was bound to a primer terminus.
dCTP and dGTP (the 3”terminal nucleotides of the primer
and the first4 nucleotides needed for synthesis) were present
during the preincubation to protect theprimer from removal
of the terminalnucleotides by the 3‘+5‘ exonuclease activity
of the polymerase (see below). After a short preincubation
-%DNA
period,
an excess (%fold over 4X ssDNA circles) of challenge
cssDNA
DNA, i.e. singly primed,M13Goril ssDNA (8.6 kb), was
FIG. 3. Time course of replication of singlyprimed &X added. After further preincubation for either 5 s or 3 min,
ssDNA analyzed by neutral agarose gel electrophoresis. Au- replication was initiated by the addition of dATP and[ c Y - ~ ~ P ]
toradiogramsof 0.8%neutral agarose gel
electrophoresis of replication dTTP; after6 min, further incorporation of radioactivity was
reactions of Fig. 1in the absence of SSB ( A )and replication reactions prevented by addition of excess unlabeled dTTP. Incubation
of Fig. 1 in the presence of SSB (B).
Electrophoresis and autoradiog- for an additional 40 min ensured complete replication of
raphy were as described under “Experimental Procedures.” The arrows mark the ,position of RF I1 DNA and singly primed ssDNA templates towhich a polymerase molecule was bound at the
time unlabeled dTTP was added. Analysis of the replication
standards detected by UV-induced ethidium bromide fluorescence.
products by agarose gel electrophoresis is shown is Fig. 4B.
in the reaction (2-3 min). However, at later times (after 5 Essentially allthe label was incorporated into the4X ssDNA
min), thepolymerase activity was maximal at 60-90 mM NaCl template following the 5-s preincubation period. Hence, the
and then decreased progressively as the salt concentration herpes DNA polymerase remained bound to the 4X DNA
was increased. Analysis of the products of replication by during synthesis.After a 3-min preincubation period, most of
neutral agarose gel electrophoresis showed that most of the the radioactivity was associated with the 4X DNA, showing
only minimal transfer of the herpes polymerase to the chalprimers were extended to a few uniquepositions on the
circular template (data notshown, but see Fig. 3A). A differ- lenging M13Goril DNA before initiation of synthesis.A
ent setof polymerase pause or termination sites
was observed controlreaction in which the polymerase was added to a
with another 15-mer (primer 4 in Ref. 22) which hybridizes mixture of $X and M13GorilssDNAs showed about twice as
to 4X ssDNA at a site 1.9 kb distant from the primer 1 15- much incorporation of labeled nucleotide into the M13Goril
$X ssDNA (Fig. 4B, third l a n e ) , consistent
mer. In contrast to thestimulation of activity in the absence ssDNA as into the
I
30
v)
v)
20
I
I
I
NaCl
HSV-1 DNA Polymerase
A)
4255
[32u-P]dTTP
DNA
'$xl
74 dCTP
G o r i ldGTP
M13
CHALLENGE
SSDNA
--
M 1 3 G o r iR
l Fll-
0x174 R F I I
FIG.4. Processive DNA replication by herpes DNA polymerase in presenceof challengingtemplate.
A , scheme of challenge experiment. Herpes DNA polymerase (7 fmol) was preincubated for 3 min (30 "C) with 120
fmol of singly primed 4X ssDNA (as circles) coated with SSB (2.2 pg) in 25 p1 of buffer A containing 60 p~ each
dCTP and dGTP.After the preincubation, the reaction was mixed with a solution (25 pl) containing 240 fmol of
singly primed M13Goril ssDNA (as circles) and 6.9 pg of SSB in buffer A containing60 p~ each dGTP anddCTP.
After further incubation for either 5 s or 3 min, 2 pl of a solution containing 1.5 mM dATP and 0.5 mM [m3*P]
dTTP (3725 cpm/pmol) was added. After 6 min, excess unlabeled dTTP was added (1 mM final concentration),
and the incubation was continued for 40 min before quenching with an equal volume of 1%SDS and 40 mM
EDTA. B, autoradiogram of 0.8% neutral agarose gel electrophoresis of replication reactions. Addition of dATP
and [ c Y - ~ ~ P I ~was
T T Peither 5 s (first lune) or 3 min (second l a n e ) after addition of Ml3Goril DNA. The third
lune (marked C ) is a control reaction where herpes DNA polymerase was added to a mixture of the @X(120 fmol)
and Ml3Goril (240 fmol) ssDNAs. The tick marks correspond to theposition of DNA standards detected by UVinduced ethidium bromide fluorescence.
with the molar excess of M13Goril over 4X ssDNA circles.
The concentrations of primed DNA circles and HSV-1
DNA polymerase in the experiments of Figs. 3 and 4 provide
an upper limit of 1 nM for the equilibrium dissociation constant (&) for polymerase binding to a primer-template. Unlike E. coli DNA polymerase I11 holoenzyme, whose tight
binding to DNA and highly processive DNA synthesis requires
ATP (or dATP) hydrolysis, there was no effect of ATP or
dAMP-PNP on processive DNA synthesis by the HSV-1 DNA
polymerase (data notshown).
HSV-1 DNA Polymerase Completely Replicates Primed 4X
ssDNA Circles-To determine the extent to
which the circular
DNA template is replicated by the herpes DNA polymerase,
the products of replication of singly primed 4X ssDNA were
treated with E. coli DNA ligase, which requires directly apposing 3'-hydroxyl and 5'-phosphoryl termini toform a phosphodiester bond (23). In the experiment shown in Fig. 5, a
portion of the reaction mixture was removed after 10 min,
and E. coli DNA ligase and NAD+ were added. Samples were
removed after an additional 10, 20, and 30 min of incubation
and analyzed by electrophoresis in an agarose gel containing
ethidium bromide (Fig. 5B, eighth to tenth lanes). Approximately 70% of the RFI1 products of polymerase action were
sealed by the DNA ligase within 30 min and asa consequence
co-migrated with a closed circular duplex DNA marker. It
therefore appears that synthesis by the herpes DNA polymerase proceeds directly to the 5' terminus of the primer,
leaving a nick that canbe sealed by DNA ligase.
HSV-1 DNA Polymerase Lacks 5 ' 4 ' Exonuclease Activity-The experiment used to test for 5'+3' exonuclease activity associated with the herpes DNA polymerase is diagramed in Fig. 5A. A 2-fold molar excess of polymerase was
added to SSB-coated 4X ssDNA primed with a synthetic 15mer labeled with 32Pa t its 5' terminus. Samplesof the reaction
were removed a t intervals up to 30 min, the time required to
replicate the entire circular template. If the polymerase has
significant 5'43' exonuclease activity, it should remove the
32P-labeled 5"terminal nucleotide of the primer upon complete replication of the template. As shown in Fig. 5B, the 5'terminal nucleotide persisted throughout the 30-min period
of replication. Quantitation of radioactivity of excised gel
slices showed that after30 min, the 32Pat theposition of the
completed RF I1 circles was approximately 75% that of the
primer before the beginning of replication (0 min); 19%of the
remaining 32Pwas present in the smear below the RF I1
products.
The reaction of Fig. 5 was initiated by adding DNA polymerase to a solution containing the primed DNA and all four
dNTPs; hence, the replication intermediates appeared as a
smear. A discreteband of replication intermediates (as inFig.
3) is observed only when the polymerase is preincubated for
a brief period with the primed DNA and synchronous replication is initiated by addition of the dNTPs.
Herpes DNA Polymerase and 3 ' 4 ' Exonuclease Are Present within the Same Polypeptide-The HSV-1 DNA polymerase and its associated 3'45' exonuclease co-sediment perfectly in a 10-30% glycerol gradient at theposition of a 158kDa marker protein (Fig. 6A), consistent with previous reports (24, 25). Moreover, as shown in Fig. 6B, the herpes
DNA polymerase is nearly homogeneous (>go%) as judged by
Coomassie Blue staining of the gradient fractions following
SDS-polyacrylamide gel electrophoresis. The DNA polymerase and 3'+5' exonuclease active sites would therefore appear
to reside within the same polypeptide chain. The herpes
polymerase showed no detectable primase activity (data not
shown).
3 ' 4 ' Exonuclease Has Proofreading Activity-An earlier
report demonstrated that the 3'+5' exonuclease associated
with the herpes DNA polymerase has proofreading activity
(26). We have examined the proofreading capacity of the 3'+
HSV-1 DNA Polymerase
4256
A)
5’ LABELLED
DNA 15-MER
V
RFIl
DNA
LIGASE
RFI
*
B)
MINUTES: 0
3 6 10 15 2030 10 20 30
=-RFlI
--RFl
(Table 11)is considered more fully in the accompanying report
(10).
During DNA synthesis with the homopolymer template,
(dA)looo. (dT),,, a significantamount of dTMP was generated,
approximately 10% of the level incorporated into the homopolymer (compare Fig. 7, B and C). However, in the absence
of aprimer-template, dTMP was not produced (data not
shown). The dTMPis most likely formed upon hydrolysis of
paired 3’ termini during DNA synthesis as observed previously for the herpes polymerase (25) and for other DNA
polymerases that contain3‘+5‘ exonuclease activity (1).
Phosphonoacetic Acid Inhibits DNA Polymerase and 3 ‘ 4 ’
Exonuclease to Equal Extents duringDNA Synthesis-Phosphonoacetic acid inhibits both the polymerase and 3’+5’
exonuclease activities of the herpes DNA polymerase with a
K j value of approximately 4 p M (Fig. 8, A and B ) . In the
absence of dNTPs, theKi for phosphonoacetic acid inhibition
of the 3’+5’ exonuclease on the hairpin template with either
paired or unpaired 3’ termini is 50 p~ (Table 11), the same as
the K , for hydrolysis of both paired and unpaired 3’-terminal
nucleotides (see above).
DISCUSSION
Despite its slow rate of DNA synthesis, the HSV-1-induced
+ssDNA
DNA polymerase is strikingly processive. Once bound to its
primer-template, the herpes polymerase does not dissociate
FIG.5. Complete replication of primed &X ssDNA circles by
herpes DNA polymerase which lacks 5‘43’ exonuclease ac- during the approximately 20 min required to fully replicate a
tivity. A , scheme for detecting 5’+3’ exonuclease activity activity 5.4-kb +X ssDNA circle. This highly processive mode of
and complete replication of ssDNA. The 5’ end-labeled 15-mer primer nucleotide polymerization may be ofimportance in replicating
annealed to @XssDNA was prepared as described under “Experimen- the 150-kb viral chromosome and, even more important, in
tal Procedures.” A slight excess of herpes DNA polymerase (1000 the synthesis of multiple copies of the genome in rolling circle
fmol) was added to initiate replication of the singly primed @XssDNA DNA replication (2). Although it is not known whether Oka(1.3 pg, 750 fmol as circles) in 150 pl of buffer A containing 13 pg of
SSB, 0.5 mM ATP, 60 p~ dCTP, dGTP, and dATP, and20 p~ [3H] zaki fragments are intermediates in replication of the herpes
removed and chromosome, the high processivity, replication to a nick sealdTTP (2500 cpm/pmol). After 10 min, 50plwas
incubated with NAD+ (0.1 mM final concentration) and 0.3 pg of E. able by DNA ligase, and lack of 5‘+3‘ exonuclease activity
coli DNA ligase.Samples (12.5 pl) were removed at thetimes indicated are clearly desirable features in thesynthesis of discontinuous
and quenched with SDS/EDTA; the DNA products were analyzed by DNA fragments.
neutral agarose gel electrophoresis; and DNA synthesis was quantiThe rateof fork movement during replication of pseudoratated as described under “Experimental Procedures.” €?,autoradibies
virus, a member of the herpes virus family, is approxiogram of 0.8% neutral agarose gel electrophoresis of the products of
replication. Eight to tenth lunes, the time noted is after the addition mately 50 nucleotides/s at 37 “C (26),similar to that of
of ligase. Arrows mark the positions of RF 11, RF I, and ssDNA eukaryotic chromosomes (27, 28). One might therefore anticstandards detected by UV-induced ethidium bromide (EtBr)fluores- ipate a turnover number of at least 30-40 nucleotides/s for
cence.
the herpes DNA polymerase at 30 “C. The apparentturnover
number of 0.25 nucleotide/s with singly primed 4X ssDNa
5’ exonuclease using defined synthetic DNA hairpin tem- circles is far too low to sustainproductive herpes virus infecplates (57-mers) having either a paired or unpaired labeled 3‘ tion (assuming 10,000 copies/cell in 10 h). However, the 20terminus (diagramed in Fig. 7A). In the presence of the 4 fold stimulation of the herpes DNA polymerase upon coating
dNTPs, the 3‘+5‘ exonuclease completely removed the un- the ssDNA with E. coli SSB approaches the ratein uiuo. HSV1 may therefore encode a functional analogue of E. coli SSB.
paired3‘-terminal nucleotide (Fig. 7B).Incontrast,the
Like
E. coli SSB, the herpes-induced ICP8 binds tightly and
paired 3‘ terminus was stable to the 3‘45’exonuclease in the
presence of dNTPs (Fig. 7B), due presumably to theaddition cooperatively to ssDNA (9), is essential for ongoing DNA
of nucleotides to the paired 3‘ terminus by the polymerase. replication (8), and is present a t stoichiometric levels (29).
However, despite its similarity to the E. coli SSB, binding of
The 3’+5’ exonuclease hydrolyzed an unpaired 3’-terminal
nucleotide at justover twice the rate atwhich the paired 3’- ICP8 to ssDNA completely inhibits the replication of singly
terminal nucleotide was hydrolyzed (Fig. 7C). The rates of primed +X ssDNA by herpes DNA polymerase (10). In conhydrolysis of paired and unpaired 3‘-terminal nucleotides did trast, ICP8markedly stimulates synthesis by the polymerase
not change over the range 1p M to 50 nM template, setting an on duplex DNA templates (10).
E. coli DNA polymerase I11 holoenzyme hydrolyzes the P,rupper limit of the K , for hydrolysis at 50 nM (Table 11). Since
substrate was present at saturating concentrations, apparent phosphodiester bond of ATP (or dATP) to initiate highly
turnover numbers for removal of paired and unpaired 3’- processive DNA synthesis (22). In contrast, the monomeric
terminal nucleotides could be calculated from the observed herpes DNA polymerase does not require ATP or dATP.
hydrolysis of the
rates of hydrolysis (Table 11). Use of Mn2+ inplace of Mg2+ Thus, a complex subunitstructureand
hadno effect on the rate of hydrolysis of the paired 3’ terminal phosphate of ATP or dATP are not essential for
terminus but stimulatedremoval of the unpaired 3’ terminus highly processive DNA synthesis.
The response of the herpes DNA polymerase to ionic
1.5-fold (Table 11). The effect of the herpes-encoded ssDNAbinding protein,ICP8,on
the 3‘+5‘ exonuclease activity strength is complex. At a molar excess of DNA polymerase to
HSV-1 DNA Polvmerase
4257
B)
-
1
I
FIG. 6. Glycerol gradient sedimentation analysis of herpes DNA
polymerase. A, glycerol gradient sedimentation of herpes DNA polymerase is
described under“Experimental Procedures.” DNA polymerase (Pol, 0) was
assayed using activated calf thymus
DNA, and the 3’+5’ exonuclease (Exo,
A) was assayed using the synthetic 57mer hairpin DNA with a labeled unpaired 3’ terminus as described under
“Experimental Procedures.” Position of
protein standards in a parallel glycerol
gradient are marked by brackets. B, SDSpolyacrylamide gel electrophoresis of
glycerol gradient peak fractions. The tick
marks correspond tothe migration of
molecular mass standards in the same
gel.
40
kda158316
E
c2
+
H
100
I
FRACTION NUMBER
20 2122
I
.”
”.
-.-
i
25
2 32 4
~-e,.
.. .
44
17
“
-,
stds
kda
-116
- 92
80
-66
y
,,
60
ee
5
2
40
YI
Q:
2
-
IQ
- 45
2o
30
20
10
FRACTION NUMBER
TABLE
I1
Steady state kinetic parameters for hydrolysis of pairedand unpaired
3’ termini by 3‘+5‘ exonuclease of HSV-1 DNA polymerase
3‘ termini
AI
3’PAIRED
7
21
A
18
3’ UNPAIRED
Unpaired
Paired
*
22
Turnover number (nucleotides/s)
30 “C, no addition
0.16
0.066
37 “C, no addition
0.23 0.073
30 “C, ICP8 added
0.046 0.178
30 “C, Mn2+
0.066
K , 3’ termini ( p ~ )
C0.05
<0.05
Ki PAA (pM)”
50
17
B)
a
0.24
50
PAA, phosphonoacetic acid.
primer termini, the polymerase is stimulated at early times of
synthesis by an ionic strength of 100 mM; however, it is
inhibited at later times under theseconditions. This behavior
may be explained by salt stimulation of a rate-limiting step
I
I
1
I
I
4
3
1
2
in polymerase catalysis while enzyme is bound to DNA, which
is offset by an increase in the stability of DNA sequencespecific pause sites at salt concentrations above 100 mM (e.g.
hairpin structures). However, at substoichiometric ratios of
polymerase to primer termini, polymerase activity is stimulated up to anionic strength of 250 mM. Under these conditions, the polymerase can extend a primer
to a sequencespecific pause site and then dissociate from the terminus to
gain access to anunused primer terminus.Maximum activity
of herpes DNA polymerase onSSB-coated singly primed
ssDNA circles at moderate ionic strength (60-70 mM) may
result from the combined effect of SSB-induced removal of
sequence-specific barriers and anintrinsic stimulation of nucleotide polymerization at thisionic strength.
The exceptionally low K,,, value of the 3‘41‘exonuclease
for a primer terminus may underlie the only 2-fold difference
in rates of removal of paired and unpaired 3‘ termini. The
1
2
3
4
3‘+5‘ exonuclease of the DNA polymerase isolated from
MINUTES
HSV-2-infected cells has been reported to remove a 3‘-unFIG. 7. Comparison of paired and unpaired 3’ termini as paired terminus six times faster than a 3’-paired terminus in
substrates for the 3’+5’ exonuclease of herpes DNA polym. ~ basis for the discrepancy between
erase. A, diagram of synthetic DNA hairpin substrates labeled at the the absence of ~ N T P sThe
that
result
and
the
one
presented here is unclear, but may
3’ terminus with either a paired deoxyadenylate or unpaired deoxyand/or source of
thymidylate residue; B, plots of 3’+5’ exonuclease activity on the reside in the differentassayconditions
3’-paired and -unpaired hairpin templatesin thepresence of dNTPs; enzyme.
C, plots of3’+5’ exonuclease activity on 3”paired and -unpaired
hairpin templates in the absence of dNTPs. Reactions were as described under “Experimental Procedures.”
J. Abbotts, Y. Nishiyama, S. Yoshida, and L. A. h e b , unpublished
observations.
HS V-1 DNA Polymerase
4258
A) DNA POLYMERASE
8 ) 3”5’ EXONUCLEASE
20
FIG. 8. Effect of phosphonoacetic
acid on herpes DNA polymerase and
3‘45‘ exonuclease during DNA
synthesis. The reaction mixture (50 pl)
contained 2.5pgof (dA)lm.(dT)l, (1:2
molar ratio), 240 fmol of DNA polymerase, 60 mM NaCl, and 20 p~ [3H]dTTP
(18.000 cpm/pmol) in buffer A. After 3
min at 30 “C, 10-pl samples were placed
in separate tubes containing1pl of HzO,
1pl of 44 p~ phosphonoacetic acid, or 1
pl of 1.1mM phosphonoacetic acid. Samples (1 pl) were removed at the times
indicated and quenched immediately
upon adsorption to a polyethyleneiminecellulose strip. Chromatography and
quantitation of dTMP and products of
synthesis (origin) were as described for
the 3 ’ 4 ’ exonuclease assay under “Experimental Procedures.” PAA, phosphonoacetic acid.
I
I
/
I
ADDITION
/“NO
5
I
1
1
10
5
MINUTES
TABLEI11
ComDarisonof HSV-1 DNADolvmerase with DNADolvmerase 01
10
MINUTES
REFERENCES
1. Kornberg, A. (1980) DNA Replication, W. H. Freeman, San
Francisco
2. Roizman, B., and Betterson, W. (1985) in Virology (Fields, B. N.,
ed) pp. 497-526, Raven Press, New York
136.000
280,000
Mr
3. Powell, K. L., and Purifoy, D. J. (1977) J. Virol. 24, 618-626
No. of subunits
i
4
4. Gibbs, J. S., Chiou, H. C., Hall, J. D., Mount, D. W., Retondo,
Primase
No
Yes
M. J., Weller, S. K., and Coen, D. M. (1985) Proc. Natl. Acad.
3‘+5’ exonuclease
No
Yes
Sci. U. S. A. 82, 7969-7973
No
No
5’+3’ exonuclease
5. Quinn, J. P., and McGeoch, D. J. (1985) Nucleic Acids Res. 13,
Rate of polymerization (nucleo2
4.5
8143-8163
tides/s/enzyme)b
6. Conley, A. J., Knipe, D. M., Jones, P. C., and Roizman, B. (1981)
15
>5000
Processivity
J. Virol. 37, 191-206
Inhibits
Stimulates
Effect of 0.2 M NaC1’
7. Weller, S. K., Lee, K. J., Sabourin, D. J., and Shaffer, P. A.
Inhibition by PAAd
No
Yes
(1983) J. Virol. 45, 354-366
DNA polymerase from Drosophila embryos (33).
8. Shaffer, P. A., Bone, D. R., and Courtney, R. J. (1976) J. Virol.
* Singly primed 4X ssDNA coated with E. coli SSB astemplate.
17,1043-1048
e Activated calf thymus DNA template.
9. Ruyechan, W. T. (1983) J. Virol. 46,661-666
PAA, phosphonoacetic acid.
10. O’Donnell, M. E., Elias, P., Funnell, B. E., and Lehman, I. R.
(1987) J. Bwl. Chem. 262,4260-4266
It is not surprisingthat t h e 3 ‘ 4 5 ‘ exonuclease and polym- 11. Eisenberg, S., Harbers, B.. Hours,. C... and Denhardt. D. T. (1975)
.
.
J. Mol.-koi. 99, 107-123
erase activities share the same polypeptide chain. Other po12. Aposhian, H. V., and Kornberg,
J. Biol. Chem. 237.
-. A. (1962)
.
lymerases with associated exonuclease activity have both ac519-525
tive sites on a single polypeptide (1). However, the roughly 13. Soltis, D. A., and Lehman, I. R. (1983) J. Bwl. Chem.258,6073equal sensitivity of the herpes polymeraseand 3‘+5‘ exonu6077
clease activities to inhibition by phosphonoacetic acid during 14. McHenry, C., and Kornberg, A. (1977)J. Biol. Chem. 252,64786484
DNA synthesis was unexpected. In the case of the E. coli
DNA polymerase I large fragment (31), crystallographic anal- 15. Panasenko, S. M., Alazard, R. J., and Lehman, I. R. (1978) J.
Bwl. Chem. 253,4590-4592
ysis has demonstrated that
the two active sitesare physically 16. Post,
L. E., Mackem, M., and Roizman, B. (1981) Cell 24, 555separated from each other by approximately 25A (32). Pos565
sibly,phosphonoaceticacidbindstooneactivesite
and 17. Elias, P., O’Donnell, M. E., Mocarski, E. S., and Lehman, I. R.
(1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6322-6326
thereby prevents switching of the primer 3‘ terminus to the
18. Alberts, B., and Herrick, G. (1970) Methods Enzymol. 23, 1908other site.
1917
The herpes polymerase differs in many important respects
19. Laemmli, U.K. (1970) Nature 227,680-685
from DNA polymerase (Y (Table 111). These differences may
20. Read, M., and Northcote, D. H. (1981) Anal. Biochem. 116,53reflect differences inthe complexity of replicating host chro64
mosomes organized into a complex nucleosomal structure as 21. Sanger, F., Coulson, A. R., Friedmann, T., Air, G. M., Barrell, B.
compared with the relatively simple 150-kb HSV-1 genome.
G., Brown, N. L., Fiddes, J. C., Hutchison, C. A., 111, Slocombe,
P. M., and Smith, M. (1978) J. Mol. Biol. 125,225-246
In both cases, however, the DNA polymerase is very likely
that could very 22. O’Donnell, M. E., and Kornberg, A. (1985) J. Bwl. Chem. 260,
associated with accessory replication proteins
12875-12883
significantly alter their catalytic properties.
23. Lehman, I. R. (1974) Science 186, 790-797
Acknowledgment-We are grateful to Ed Mocarski for expert ad- 24. Weissbach, A., Hong, S.-C. H., Aucker, J., and Muller, R. (1973)
J. Biol. Chem. 248,6270-6277
vice and assistance in the handling of cells and viruses.
HSV-1 DNA
DNA
polymerase
polymerase a’
HSV-1 DNA Polymerase
25. Knouf. K.-W. (1979) Eur. J. Biochem. 98. 231-244
30.
26. BenlPorat, T.,Blankenship, M. L., DeMarchi, J. M., and Kaplan,
A. S. (1977) J. Virol. 2 2 , 734-741
27. Blumenthal, A. B., Kriegstein, H. J., and Hogness, D. S. (1973)
Cold Spring Harbor Symp. Qwnt.Bwl. 3 8 , 205-22332.
28. Huberman, J. A., and Riggs, A. D. (1968) J. Mol. Biol. 32, 327-
4259
Deleted proof
in
31. Setlow, P., Brutlag, D., and Kornberg, A. (1972) J. Biol. Chem.
247,224-231
Ollis, D. L., Brick, P., Hamlin, R., Xhong, N. G., and Steitz, T.
A. (1985) Nature 313, 762-766