ANDREW M. HOLMES,* STEVEN M. WIETSTOCK, AND WILLIAM T. RUYECHAN. Department of Biochemistry, Uniformed Services University of the Health ...
Vol. 62, No. 3
JOURNAL OF VIROLOGY, Mar. 1988, p. 1038-1045
0022-538X/88/031038-08$02.00/0 Copyright © 1988, American Society for Microbiology
Identification and Characterization of a DNA Primase Activity Present in Herpes Simplex Virus Type 1-Infected HeLa Cells ANDREW M. HOLMES,* STEVEN M. WIETSTOCK, AND WILLIAM T. RUYECHAN
Department
of Biochemistry,
Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 208144799 Received 31 July 1987/Accepted 1 December 1987
A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. In contrast, the pH optimum of the HeLa DNA primase was very sharp and fell between pH 7.9 and 8.2. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase and was eluted from single-stranded DNA agarose at higher salt concentrations than the host primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4°C for several weeks, the DNA primase separated from the viral DNA polymerase. Separation or decoupling could also be achieved by gel filtration of the HSV polymerase:primase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, we believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA.
mammalian cells contain oligoribonucleotides of this size at their 5' ends (29, 33). Mature HSV DNA molecules contain alkali-labile sites (12, 13, 31, 36). These may be due to the use of RNA primers for DNA replication, some of which may have persisted during maturation of the DNA, although alternative explanations for alkali lability and the presence of RNA-like material in mature viral DNA are possible (3, 25, 38). Newly synthesized HSV DNA has been demonstrated to contain RNA covalently linked to DNA (2, 15, 24) which is removed over a period of time (24). These RNA segments are estimated to be 35 nucleotides in length and are linked to DNA averaging 36 x 103 nucleotides in length (24). This would indicate a major difference between the way the virus uses RNA to prime DNA synthesis and the way eucaryotic cells replicate. The length of the DNA attached to RNA primers in the eucaryotic cell is usually 200 nucleotides or less (8). These differences may reflect the involvement of viruscoded proteins in the synthesis of the RNA primers as well as in the synthesis of the viral DNA. We report here on the identification and characterization of an enzyme which can be classified as a DNA primase from HSV-1-infected HeLa cells and whose enzymatic and structural properties distinguish it from the DNA primases isolated from uninfected HeLa cells.
The isolation of a virus encoded DNA polymerase from herpes simplex type 1 (HSV-1)-infected cells (21, 22, 35) was an important first step toward understanding viral DNA replication. The DNA polymerases isolated from HSV-1and HSV-2-infected cells show very similar properties (26) which serve to distinguish the viral enzyme from mammalian DNA polymerases. These properties include salt optimum, inhibition by polyamines (such as spermine), inhibition by phosphonoacetate and phosphonoacetate analogs, column chromatographic behavior, sedimentation rate on sucrose gradients, and inhibition by 9-(2-hydroxyethoxymethyl)GTP and 9-(1,3-dihydroxy-2-propoxymethyl)GTP (10, 11, 26). The presence of
a
3'
->
5' exonuclease
activity (9, 26, 35)
also serves to distinguish HSV DNA polymerase from mammalian DNA polymerases ox, and -y. The size of HSV DNA polymerase was originally estimated to be 180 kilodaltons (kDa) (35), but a polypeptide with a molecular size of approximately 140 kDa has now been assigned to the DNA polymerase activity (26), and the gene for this polypeptide has been cloned and sequenced (28). Although the properties of HSV DNA polymerase distinguish it from mammalian DNA polymerases, one important similarity exists between HSV DNA polymerase and mammalian DNA polymerase a., the enzyme believed to be responsible for the bulk of the cellular DNA replication (see reference 16 for review). That property is the ability to use a short piece of RNA as a primer for DNA synthesis (35). A DNA primase can be copurified from eucaryotic cells in tight association with DNA polymerase a (6, 14, 20, 34). This primase can synthesize oligoribonucleotides of 8 to 10 bases in length, which the DNA polymerase can then use in DNA synthesis (7, 17, 32). Nascent DNA chains extracted from 3,
*
MATERIALS AND METHODS
Chemicals and reagents. Calf thymus DNA and unlabeled deoxynucleoside and nucleoside triphosphates were obtained from Sigma Chemical Co. [8-3H]dATP, [methyl3H]TTP, and [a-32P]ATP were obtained from New England Nuclear Corp. Escherichia coli DNA polymerase I was obtained from New England BioLabs, Inc. Sepharose 6B was obtained from Pharmacia Fine Chemicals. Single-
Corresponding author. 1038
VOL. 62, 1988
stranded DNA agarose was obtained from Bethesda Research Laboratories. Tissue culture media were obtained from GIBCO Laboratories. Poly(dT)3500 was prepared by using terminal deoxynucleotidyltransferase as described elsewhere (5). Calf thymus DNA polymerase:primase complex and free calf thymus DNA primase were prepared as described previously (6). Antibodies were prepared by immunizing individual rabbits with individual peptides from the calf thymus DNA polymerase:primase complex as described elsewhere (18). They were further purified by chromatography on protein A-Sepharose and then affinity purified by chromatography on a calf thymus DNA polymerase:primase complex Sepharose 4B column. All other chemicals were of reagent grade. Cells and viruses. HeLa S-600 cells were grown in suspension culture in suspension-modified Eagle medium to a concentration of 1.6 x 106/ml. Infection was with HSV-1 (mP) grown in Vero cells at a multiplicity of 20 PFU per cell as described by Derse et al. (10). Cells were harvested 8 h after infection, the time viral DNA replication reached its maximum under these conditions. The cells were harvested by centrifugation and stored at -70°C or used immediately for the isolation of HSV DNA polymerase. Isolation of HSV DNA polymerase:primase. The isolation procedure was based on a procedure described by Powell and Purifoy (27) and Ruyechan and Weir (30). The cells were suspended in extraction buffer (20 mM Tris [pH 7.5], 1.7 M KCI, 5 mM EDTA, 1 mM dithiothreitol [DTT], 0.2% Nonidet P-40, 10% glycerol), sonicated three times for 30 s in a Heat Systems Ultrasonics sonicator, and centrifuged at 500 x g, and the supernatant was subjected to a 10% polyethylene glycol phase separation. The precipitated material, including nucleic acids, was spun down. The supernatant was dialyzed against 0.15 M KCI in the buffer described above and chromatographed on a single-stranded DNA agarose column. Enzyme were eluted with a linear 0.15 to 1.0 M KCI gradient. All procedures were carried out at 4°C in a solution containing 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM DTT, and 10% (vol/vol) glycerol. A modified procedure of Allen et al. (1) has also been used to isolate DNA polymerase from HSV-1-infected HeLa cells. In this procedure, the cells were lysed in hypotonic buffer, brought to 0.4 M potassium phosphate, as described above, and passed through DEAETris-acryl. The unbound material was dialyzed against a solution containing 20 mM potassium phosphate (pH 7.5), 1 mM DTT, 20% (vol/vol) glycerol, and 0.2% Nonidet P-40 and rechromatographed on DEAE-Tris-acryl. HSV DNA polymerase bound to the column was eluted with a linear KCI gradient (0 to 0.5 M). Fractions containing DNA polymerase:primase assayable at 0.2 M salt were pooled, dialyzed against the above buffer, and chromatographed on a single-stranded DNA agarose column under identical elution conditions. In some instances, the DNA polymerase:primase was concentrated by adsorption and batch elution with 0.5 M KCI from a 1-ml single-stranded DNA agarose column and stored at 4°C. Enzyme assays. DNA polymerase activities were assayed by using three different protocols. DNA polymerase eluted from single-stranded DNA agarose columns to which highsalt extracts had been applied were carried out in a solution containing 50 mM HEPES (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid; pH 8.0), 8 mM MgCl,, 300 pg of DNase I-treated DNA per ml, 0.2 mM each dATP, dCTP, and dGTP, 0.04 mM [3H]TTP at 372 dpm/pmol of 2 mM 2-mercaptoethanol, and 200 mM KCI. Aliquots (10 LI) of
HSV DNA PRIMASE
1039
individual column fractions were added to 40 ,ul of the reaction mixture and incubated at 35°C for 20 min. Aliquots (40 IL) were spotted on glass fiber squares and placed in 5% trichloroacetic acid-1% sodium pyrophosphate. The squares were washed twice with 1 N HCl and once with 95% ethanol, air dried, and counted in a liquid scintillation counter. One unit of DNA polymerase activity in this assay is defined as the incorporation of 1 pmol of [3H]TMP into insoluble material in 1 min at 35°C. DNA polymerase isolated by the modified procedure of Allen et al. (1) was assayed in 40 mM phosphate with or without 200 mM KCl. All other conditions were exactly as described above. After the initial isolation of HSV DNA polymerase activity by these two methods, all additional polymerase assays were carried out in a solution containing 40 mM potassium phosphate (pH 7.0), 8 mM MgCl2, 100 ,ug of bovine serum albumin (BSA) per ml, 1 mM DTT, 100 ,ug of DNAase I-treated DNA per ml, 0.1 mM each dATP, dCTP, and dGTP, and 0.1 mM [3H]TTP at 40 to 50 cpm/pmol, with or without 0.15 M NaCl. Assays were incubated at 35°C, and at the times indicated, samples were spotted on glass fiber squares and placed in the 5% trichloroacetic acid-1% sodium pyrophosphate. One unit of DNA polymerase activity is defined as the incorporation of 1 nmol of [3H]TMP into acid-insoluble material in 60 min at 35°C in this assay. DNA primase was assayed directly in a solution containing 50 mM Tris (pH 8.0), 8 mM MgCl2, 100 ,ug of BSA per ml, 1 mM DTT, 10 ,uM poly(dT)3,500, and 0.1 mM [a32P]ATP (100 to 200 cpm/pmol). Assays were done at 35°C, and aliquots were spotted on Whatman DE-81 papers previously spotted with an equal volume of 5 mM unlabeled ATP. This technique reduces contamination by the [32P]ATP. The papers were washed four times with 0.2 M dipotassium hydrogen phosphate and twice with glass-distilled water and counted in glass-distilled water in a liquid scintillation counter set on the 3H-labeled channel. The counts remaining on the paper give a direct measurement of oligoribonucleotide synthesis. One unit of enzyme activity is defined as the incorporation of 1 nmol of [32P]AMP into oligoribonucleotide in 60 min at 35°C. DNA primase was routinely assayed by an indirect assay. The assay conditions were as described above, except that 2 mM unlabeled ATP replaced the 0.1 mM [at-32P]ATP and 0.1 mM [3H]dATP at 40 to 50 cpm/pmol and 20 U of E. coli DNA polymerase I per ml was usually present. Assays were carried out at 35°C, and aliquots were spotted on glass fiber paper and processed as for DNA polymerase assays. This coupled assay procedure measures the extension of primers synthesized by the DNA primase, and the rate of incorporation of [3H]dAMP into acid-insoluble product is dependent on the rate of primer synthesis. In the absence of ATP or DNA primase, no incorporation of [3H]dAMP is observed. This is an amplification of DNA primase activity. One unit of enzyme activity in this instance is defined as the incorporation of 1 nmol of [3H]dAMP into acid-insoluble material in 60 min at 35°C. Exonuclease activity was measured in a solution containing 50 mM Tris (pH 8.0), 8 mM MgCI2, 1 mM DTT, 100 ,ug of BSA per ml, and 10 ,ug of single-stranded "4C-labeled T7 DNA at 30 x 103 cpm/,ug. Assays were incubated at 35°C, and at the times indicated, aliquots were placed on glass fiber squares and the squares were processed as for DNA polymerase assays. Sepharose 6B chromatography. Sepharose 6B chromatography was carried out on an 85-by-1.4-cm column in a solution containing 0.25 M NaCl, 50 mM Tris (pH 8.0), 100 ,ug of BSA per ml, 1 mM DTT, and 20% (vol/vol) glycerol.
1040
HOLMES
ET
J. VIROL.
AL.
Fractions (50 drops, or 1.7 ml, column was calibrated with calf catalase, rabbit IgG, E. coli DNA cytochrome c, and DTT. Antibody-binding experiments.
each)
assay
were
could be reduced
ATPase affected the
thymus
polymerase
so that
assay. The
neither
incorporation
shown in Fig. 1 was taken from individual fractions in which the linear with time. The result shown in position of elution of the DNA primase the contaminating enzyme activities. primase elution position was toward polymerase elution position. The primase activity were pooled, and referred to as HSV DNA Uninfected HeLa cells were subjected tion and chromatography procedures. activity present in control extracts agarose column. This DNA polymerase ited by 0.15 M NaCI in the assay in the absence of salt. This salt-inhibited time
I,
incorporation
Antibody-binding
were carried out as follows. A enzyme preparation and 20 ,ul of rabbit (IgG) raised against a specific calf peptide (68 kDa, 55 kDa, or 48 kDa) in Tris, 0.15M NaCl, 2 mg of BSA per incubated for 18 h at 4°C. Samples
ments
Fig.
p.l
total
was
in
the
RI
added
pooled
primase.
plI
50%
TBS
h.
That
fractions
TBS
ml)
removed, and to each sample (vol/vol) protein A-Sepharose continued for an additional 18 spun down in an Eppendorf centrifuge, the resulting supernatants were polymerase activity as described
1
was
the
thymus
did
The
5-pLI
and
assayed
above.
was
also present in the
DNA
to
could
agarose
column
properties
Association of DNA primase with When extracts of HSV-1-infected graphed on single-stranded DNA polymerase activity eluted between With the indirect, or coupled, DNA DNA primase activity was detected HSV DNA polymerase peak (Fig. primase assay also detected some this area of the elution profile difficult to quantitate the DNA fractions by the direct procedure, [32_p]AMP incorporation, it was necessary assay time far beyond that shown of high levels of exonuclease activity at later times. The direct assay presence of a DNA-dependent ATPase H activity in this area of the elution The direct DNA primase assay whereas the indirect, or coupled, ATP, helping to negate the effects ATPase. Further, the coupled DNA the DNA primase activity to such and the amount of the DNA agarose HSV
the
HeLa
from
0.3
M
primase
DNA
primase
and
amount
since
profile
the
Fig.
same
although
destroyed
encoded.
By using the modified procedure isolate DNA polymerase from infected
also
of
tion was achieved between the HeLa the HSV DNA polymerase by KCI second DEAB-Tris-acryl column. polymerase was pooled, dialyzed, stranded DNA agarose as indicated ods. The elution profile from this shown in Fig. 2. The salt-sensitive eluted between 0.1 and 0.15 M KCI. DNA polymerase again eluted between Two peaks of DNA primase activity major peak was associated with cx and the minor peak eluted at peak. The first peak was pooled for enzyme comparison as the HeLa DNA primase. When this second purification uninfected HeLa cells, a salt-sensitive
profile
DNA
contained
the
assay
The
of
and
primase
in
extent
DNA
HeLa
The
500 100
the
80
rear
of
DNA
studies
300
200
40
100
1-20
procedure
activity was observed again KCI. Salt-activated DNA not observed in this elution profile peak of DNA primase activity elution position as the DNA polymerase, of DNA primase smeared off concentration of about 0.4 M KCI. 10% of the amount of enzyme infected cell extracts were subjected RNA polymerase was not detected
merase
E
.......
.20
eluting
0.15 M
25
polymerase
(data
0l 0
was
0
5
15
10
20
25
FRACTION NUMBER
the
observed,
column
Again,
FIG. 1. Chromatography of HSV stranded DNA agarose. HSV DNA
DNA
activity
polymerase
infected cells by the modified Powell Assays were done as described in DNA polymerase in the presence primase in the coupled assay I polymerase ; A, exonuclease;
and
O,
with
Materials
in
the
0,
coli
procedure.
Initial characterization relationships between the
of
the
different
HSV
DNA
ot
salt-activated
HeLa
the
polymerasee
C2
=c
shown).
small
(data
an
(data
agarose
1).
was
KCl
10%
toward
in
of
KCl
column,
agarose,
=9 C1
the
not
of the infected cell extracts. The polymerase are consistent with those polymeraseao. On application of control (uninfected) DNA agarose of DNA primase began to elute continued to elute up to about 0.4 This material, comprising less than eluting from the infected cell DNA pooled for enzyme comparison purposes control DNA primase. This relatively primase activity identified in uninfected suggest that the much greater observed in this area of the elution infected cells were subjected to consequence of the viral infection, that it is necessarily virally
RESULTS
400
[3H]dAMP
courses
KCI.
1041
HSV DNA PRIMASE
VOL. 62, 1988
TABLE 1. Effects of the addition of 0.15 M NaCl on the activities of HeLa DNA primase and freshly prepared HSV DNA primase 20
Picomoles of [3HldAMP incorporated per 5 ,ul per 60 min
0 23a 1
~100 501CD~~I z
.
EI
50 -25
la.2 ChoaorpyofHVDAplmeaeo meras in~ the 0 prsnc10 f0.MNCl 20
aige
infected and uninfected cell extracts were explored. The effect of increasing concentrations of NaCl on the HSV and HeLa DNA primases was determined by using the coupled assay in the presence of E. coli DNA polymerase I (Fig. 3). It is important to note that these primase samples also contained HSV and HeLa DNA polymerases, respectively. Freshly prepared HSV primase was less sensitive than the HeLa DNA primase, which showed the same salt sensitivity as the DNA primase activity of the calf thymus DNA polymerase:primase complex. E. coli DNA polymerase I was active throughout the range of salt concentrations used. Since HSV DNA polymerase is a salt-activated enzyme (26, 35), however, the relative salt resistance of the HSV DNA primase could have been due to the HSV polymerase beginning to function at high salt concentrations to efficiently elongate a reduced number of primers synthesized by a salt-sensitive DNA primase. Accordingly, a comparison was made of DNA primase activities in the presence and absence of 0.15 M NaCl and with and without the addition of E. coli
100 21-
75
5'
No NaCl
0.15 M NaCl
No NaCl
0.15 M NaCl
55 180
1 72
25 105
0 33
DNA polymerase (Table 1). In the absence of both E. coli DNA polymerase I and NaCI, the elongation of primers synthesized by both the HeLa and HSV DNA primases by the respective DNA polymerases was relatively efficient compared with the elongation of the primers in the presence of E. coli DNA polymerase I (Table 1). At 0.15 M NaCl, there was no incorporation of [3H]dAMP with the 14eLa DNA primase either in the presence or absence of E. coli DNA polymerase I, whereas there was still incorporation in both instances with the HSV DNA primase. Table 2 shows the activities of the DNA polymerase at the two salt concentrations. These results were obtained by using freshly prepared HSV DNA primase. The salt profile shown in Fig. 3 was obtained with HSV DNA primase which had been isolated a few days before. HSV DNA primase stored at 4°C for several weeks was almost as salt sensitive as the HeLa and calf DNA primases were (Table 3). Furthermore, although both the DNA polymerase and DNA primase activities of this stored enzyme sample had not declined significantly (the DNA primase activity being measured in the presence of E. coli DNA polymerase I by the indirect assay), the HSV DNA polymerase could now not elongate the primers produced by the DNA primase in either the presence or absence of 0. 15 M NaCl, indicating a major change in the relationship of the DNA polymerase and DNA primase in this sample. This change could be the decoupling of the two enzyme activities or the loss of another factor which mediates the two activities. This change in the salt sensitivity and the inability of the HSV DNA polymerase to function in the coupled DNA primase assay has been observed on long-term storage of the enzyme at 4°C or after dialysis of the enzyme against 50% (vol/vol) glycerol and storage of the enzyme at -20°C. Thus, these data indicate that the relative insensitivity to salt displayed by freshly prepared HSV DNA primase is not an intrinsic property of the primase but may also indicate its participation in a complex. The uncoupled HSV DNA polymerase is still capable of incorporating [3H]dAMP into product by using a poly(dT) template when given an
