VIROLOGY
77,
56-66
(1977)
Further Characterization BEATRIZ Department
of Cytobiology,
of Deoxyribonucleases G. T. POG02
The Public New
Health York,
Accepted
AND
MICHAEL
Research New York October
Institute 10016
from Vaccinia
Virus’
T. O’SHEA of The
City
of New
York,
Inc.,
27,1976
Further characterization was made of DNases present in vaccinia virus. The nature of the enzymes as they occur within cores and following solubilization was determined. Two activities were identified hydrolyzing single-stranded (ss) DNA but at exclusively a pH optimum of 4.5 or 7.8, respectively. Neither activity has any requirement for ions or cofactors. The pH 7.8 enzyme was activated preferentially by heating cores to 50”. Analysis of the products of hydrolysis by means of DEAE-paper chromatography confirmed that the pH 4.5 activity was an exonuclease and the pH 7.8 enzyme an endonuclease. The exonuclease could act on the 5’-terminus of the DNA. Both nucleases could hydrolyze poly(dT), poly(dA), and poly(dC1 to a varying degree but had no effect on poly(dG). Since the oligonucleotides arising as a product of endonuclease action did not serve as a substrate for the exonuclease, it is concluded that the two enzymes probably do not act in concert. Solubilization of both DNases was achieved by treatment of cores with salts or urea. With 0.5 M NaCl, most of the pH 4.5 activity but only lo-20% of the pH 7.8 was released. The presence of 6-8 M urea caused the solubilization of both enzymes. When in their soluble state, the nucleases could be separated by means of isoelectric focusing in either gel or liquid milieu and retain the exo- or endonucleolytic activities. The pH 4.5 DNase had an isoelectric point or p1 of approximately 4.5, and the pH 7.8 DNase had a p1 of approximately 3.7. Each activity was contained in a single protein. Further analysis of the isolated enzymes, using sodium dodecyl sulfate (SDS)polyacrylsmide-gel electrophoresis, revealed each to be a polypeptide of MW 50,000. Taken together, the evidence indicates that vaccinia cores contain two DNases with indenendent modes of action associated with separate proteins of similar molecular weights but different ~1s.
Pogo and Dales (1969a). Subsequently, other poxviruses were shown to possess similar enzymes, including rabbit pox (Aubertin and McAuslan, 1972), an insect pox (Pago et al., 19711, and Yaba monkey virus (Schwartz and Dales, 1971). The assumption that these are two independent enzyme activities was based on a demonstration of two optimum pHs of approximately 5 and 7.8, differences in substrate affinity (K,,), and the fact that the site of action is either exo- or endonucleolytic. Despite these criteria showing two activities, Rosemond-Hornbeak et al. (1974) could purify only one, the acid deoxyribonuclease activity, from vaccinia cores. Relatively little activity at pH 7.8 was reported. The purified enzyme of RosemondHornbeak et al. (1974) possessed both exo-
INTRODUCTION
Infection of cells by poxvirus elicits the induction of several deoxyribonuclease activities (Hanafusa, 1961; Jungwirth and Joklik, 1965; McAuslan, 1965; McAuslan and Kates, 1966, 1967). At least three activities have been described and differentiated according to substrate specificity, optimum pH for activity, site of action, and time of induction during the virus cycle (McAuslan and Kates, 1966; Pogo and Dales, 196913, 1971). The presence in cores from highly purified preparations of vaccinia virus of two of these nucleases, both specific for singlestranded (ss) DNA, was first reported by 1 This work was supported in part by Health Service Grant AI-12188. 2 Address reprint requests to Dr. Pogo.
Public
56 Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0042-6822
DEOXYRIBONUCLEASES
and endonucleolytic activities and had an estimated molecule weight of 105,000. However, in sodium dodecyl sulfate (SDS) gels the enzyme showed a single polypeptide of MW 50,000, suggesting that the protein is composed of two identical subunits. The discrepancy between previous fmdings and those of Rosemond-Hornbeak et al. (1974) led to the present investigation, intended to establish whether the DNases occur in or&y a single or in two polypeptides of the virus cores. MATERZALS
AND
METHODS
The IHDW strain of vaccinia virus and L, cells were used in accordance with described experimental procedures (Dales, 1963). Methods for the purification of virus particles, preparation of stripped virus and cores, as well as the assays for the nuclease activities have been published (Pago and Dales, 1969a). Solubilization of virion nucleases was obtained by the following procedure: Core preparations containing approximately 50 kg of viral proteins were resuspended in 0.5 ml of 0.1 M Tris-HCl buffer, pH 7.4, with 0.25% P-mercaptoethanol and increasing concentrations of NaCl (0.05 to 1 M) or urea (1 to 8 M). The mixture, kept at 4” for 30 min, was intermittently agitated on a Vortex mixer. The preparations were then spun for 60 min at 35,000 rpm in an SW 65 rotor employing adaptors for 0.5ml nitrocellulose tubes. The pellets obtained were then resuspended in 0.2 ml of 0.01 M Tris-HCl buffer, pH 7.2, and the amount of protein was determined by absorbance at 260 nm. Nuclease activities were measured in both the final pellet and the supernatant fractions. Separation of the virus nucleases by isoelectric focusing was carried out in a liquid system as described by Mandel(1971) or in gels, following the method of Merz et al. (1972). The supernatant samples were extensively dialyzed against 0.01 M TrisHCl buffer, pH 7.2, to remove salts or urea prior to isoelectric focusing. Discontinuous polyacrylamide-gel electrophoresis was performed as described by Obijeski et al. (1973) using as molecular weight standards bovine serum albumin
FROM
VACCINIA
VIRUS
57
(MW 68,000), ovalbumin (MW 45,000), chymotrypsin (MW 25,000>, and cytochrome c (MW 12,000). Products of degradation of the nuclease reactions were characterized as to size by means of paper chromatography (Furlong, 1965). Aliquots of the nuclease reaction mixture were applied to 28cm sheets of Whatman DE81 paper and chromatographed for 4 hr with 1 M NH,HCO,, pH 7.9, and lop3 M EDTA as solvent. The distribution of counts per minute was determined on uniform pieces cut from the chromatogram. Thymidine 5’-triphosphate (TI’P), deoxyadenosine 5’-triphosphate (dATP), and a decanucleotide of ‘M’P were used as markers. Elution of the oligonucleotides from the paper was achieved by incubation with 1 M NH,HCO, for 18 hr at room temperature. Chemicals, isotopes, and enzymes. Thymidine 5’-triphosphate and deoxyadenosine 5’-triphosphate were purchased from Sigma. A decanucleotide of ‘M’P, poly(dC), and poly(dG), as well as labeled 13Hlpoly(dT) (sp act, 12 &i/pm01 of P) and 13Hlpoly(dA> (sp act, 31.4 &i/pmol of P), were obtained from Miles Laboratories. [r32PlATP (sp act, 10.3 Ci/mmol), [814CldATP (sp act, 52.9 mCi/mmol), [53HldTP (sp act, 22.9 Cilmmol), and [83H]dGTP (sp act, 9.4 Ci/mmol) were purchased from New England Nuclear. Calf thymus DNA (from Worthington Co.) with internal and external 5’-32P-labeled termini was prepared following the procedure described by Weiss et al. (1968). For these experiments, pancreatic DNase and alkaline phosphatase from Worthington Biochemical Corp. and polynucleotide kinase from Miles Laboratories were employed. Polynucleotides with 14C-labeled 3’-OH termini were prepared by the action of terminal transferase (from Miles Laboratories) on a [3Hlpoly(dT> or [3Hlpoly(dA) primer. The conditions for this reaction were those described by Chang and Bollum (1971). For the isoelectric focusing experiments, Bio-Lyte ampholytes (Bio-Rad) were employed. 3H-labeled Bacillus subtilis DNA was a generous gift from Dr. David Dubnau of the Department of Microbiology, Public Health Research Institute .
58
POGO
AND
O’SHEA
RESULTS
a. Study of the Products ofDegradation Core Nucleuses
EXONUCLEASE
of
Previous data concerning the presence of exo- and endonucleolytic activities in poxviruses were based on an analysis of the size of degradation products, using acid precipitation, membrane or gel filtration, and sucrose gradients. In the current experiments, ion-exchange paper chromatography, capable of separating polynucleotides according to their chain length, was
Ill
It
I I,.60mm I I I 1
8.000
4,000
h
II
A
2
6
IO
Fractton
II
2
IO
Fraction
14
18
22
FIG. 2. Products of hydrolysis by the exonuclease as a function of reaction time. E. coli exonuclease III, 25 activity units, was incubated with 10 pg of sonicated “H-labeled B. subtilis DNA (sp act, 7000 cpm/pg) at 37” in a l-ml solution containing 0.06 M Tris-HCl, pH 8, 6.6 n&f MgC&, and 1 m&f p-mercaptoethanol. At 15 and 60 min, samples were withdrawn and chromatographed as indicated in Fig. 1.
3H DNA
6
14
Number
IS
22
Number
FIG. 1. Products of hydrolysis by DNase as a function of reaction time. Pancreatic DNase (2130 U/mgl, 30 pg, was incubated at 37” with 10 Fg of 3HIabeledB. subtilis DNA (sp act, 7000 cpm/pgl in 0.5ml solutions containing 0.05 M Tris-HCl, pH 7.8, and 5 mM MgCl,. At 30 and 60 min of incubation, samples were withdrawn and applied to Whatman DE81 paper and chromatographed with 1 M NH,HCO,, pH 7.9, and 10e3 M EDTA for 4 hr. The radioactivity was determined on pieces cut from the chromamgram. The arrows indicate the migration of DNA, a decanucleotide of TMP, and TTP (left to right).
used to analyze the products more directly. Two well-known nucleases were employed for comparative purposes. Labeled DNA was incubated with pancreatic DNase, an endonuclease, or with Escherichiu coli exonuclease III, and the products of hydrolysis were sampled for assay by paper chromatography at intervals during incubation. It is evident in Fig. 1 that, after action of the endonuclease for 30 min, the DNA products had sizes ranging from that of large molecules to 10 nucleotides in length. By 60 min, the only products were short-length polynucleotides and mononucleotides. In contrast, in the case of exonuclease, the size distribution of the products was quite different (Fig. 2); only mononucleotides were observed at 15 or 60 min of incubation.
DEOXYRIBONUCLEASES 1 pti
FROM VACCINIA 1
I
15mln
7 6
10.000
pH4
I
5
400 A
21,500 b 1,800 6:
59
VIRUS
h
30 mln
x
:I, 1I 1
l0,000
d
500. I” \
300
100 7mL
I
I ,x. ,x-i x .
4
8
I I2
I6
J
(/
t
‘r-a 20
Fraction
Number
FIG. 3. Products of hydrolysis by the core-associated DNases as a function of reaction time. Vaccinia cores, 10 pg, were incubated at 37” with 5 pg of denatured 3H-labeledB. subtilis DNA (sp act, 7000 cpm/pg) in 0.2-ml solut.ions containing either 0.05 M Tris-HCl buffer, pH 7.8, or 0.05 M Na-acetate buffer, pH 4.5, both with 0.5% NP40 and 0.25% P-mercaptoethanol. After 15, 30, and 60 min, aliquots were withdrawn and subjected to chromatography as indicated in Fig. 1.
When vaccinia cores were incubated with single-strand-labeled DNA at one or the other of two optimum pHs, the products of hydrolysis accumulating as a function of time were predominantly mono- or dinucleotides in the case of the pH 4.5 reaction, regardless of the time of incubation (Fig. 3); but the reaction at pH 7.8 yielded mostly oligonucleotides, although mono- or dinucleotides were also produced as a function of time (Fig. 2); suggesting processive endonucleolytic activity. These
results clearly indicate that at acid pH the core-associated nuclease activity is exclusively exonucleolytic and at pH 7.8 predominantly endonucleolytic. b. Site of Action
and Substrate
Specificity
DNase-treated calf thymus DNA labeled internally and externally at the 5’-termini with 32P, synthetic homopolydeoxyribonucleotides, and deoxyribopolynucleotides labeled at the 3’-OH ends were used as sub-
60
POGO
AND
strates for the core nucleases. When cores were incubated with appropriately labeled calf thymus ssDNA, it was found that at pH 4.5 there was a release of 32P label, indicating that the exonuclease functions at the Y-terminus. In contrast, there was no release of the 32P label at pH 7.8 (Table 11, indicating that the substrate lacked specific sequences for the endonucleolytic attack. When cores were incubated at pH 4.5 with [3H]poly(dA) as the substrate, in which the 3’-OH end was labeled with 14C, there was no release of the 14C label, indicating that the exonuclease does not attack the 3’OH end (not shown). When homopolydeoxyribonucleotides were used as the substrates, the data summarized in Table 2 showed that the pH 4.5 activity caused hydrolysis of more poly(dT) than poly(dC) and least of poly(dA) but failed to act on poly(dG). A similar order of substrate preference was shown at pH 7.8. To find out whether core nucleases work in a coordinate manner, oligonucleotides, produced by the action of the endonucleolytic cleavage, were used as substrates for the exonuclease. The results of four independent experiments indicated that the exonuclease failed to act on the products of the endonuclease reaction, suggesting that the two activities are not coordinate. This may also imply that the products of endoTABLE SITE
OF ACTION
ON ssDNA
Additions
Buffer, Buffer, Buffer, Buffer,
pH 4.5 pH 4.5, pH 7.8 pH 7.8,
1 BY THE
CORE
DNASES
[32P15’-DNA
+ cores + cores
(cpm)”
(% cpm added)”
9000.0 4850.0 8000.0 8650.0
90.0 48.5 80.0 86.5
o Expressed as acid-precipitable counts per minute. Aliquots of denatured calf thymus DNA carrying $*P-labeled 5’-termini (10,000 cpm) were incubated with 5 pg of cores in a buffered solution containing 0.5% NP40 and 0.25% P-mercaptoethanol. The reaction, allowed to occur for 60 min at 37”, was arrested by the addition of cold 10% trichloroacetic acid and 50 pg of calf thymus DNA as carrier. The precipitates formed were collected on Millipore filters for scintillation counting.
O’SHEA TABLE SUBSTRATE
2
SPECIFICITY
Nucleotide
OF CORE
DNASES
Nucleotide hydrolyzed (nmol/ mg of protein in 60 min)” pH 4.5
pH
7.8
Poly(dA) Poly(dC) Poly(dG)
1200.0 1500.0
650.0 1220.0 -
Poly(dT)
2000.0
1340.0
u [Methyl-“Hlpolydeoxythymidylate (sp act, 12 &ilpmol of P), 12.5 pg; 7 pg of [WHlpolydeoxyadenylic acid (sp act, 31.4 PCilmol of P); 1 A,,, unit of polydeoxycytidylate and 1 A,,, unit of polydeoxyguanylate were incubated for 1 hr at 37” with 5 kg of cores in buffered solutions containing 0.5% NP40 and 0.25% @-mercaptoethanol. The quantity of each polynucleotide hydrolyzed was determined by the amount of acid-soluble counts per minute released or by absorbance at 273 nm in the case of poly(dC) and at 252 nm using poly(dG).
Stripped VIWS ,E 150 E $
CWOC __-’ -1
400
1_____ I:- -----.
.f5 100 I’ h ,’ P ,’ 2 50 I
---*
,’
__--
\
,’
*&.----K-
300
I’
’
,’ 200
5
loa
s Ti0
IO
m
30
40
0
IO
20
3c
40
Minutes ot 50°C
FIG. 4. Effect of elevated temperature on vaccinia DNase activities. Stripped virus or core preparations containing 5 pg of protein in 0.2 ml of 0.01 M Tris-HCl buffer, pH 7.3, and 0.25% P-mercaptoethano1 were heated to 50” for 10, 20, and 40 min, then cooled to 4”. Afterwards, DNase activities were determined in each preparation as described in Materials and Methods.
nucleolytic cleavage have 5’-hydroxyls 3’-phosphates. c. Effect of Elevated &ease Activities
Temperature
and
on Nu-
Attempts to differentiate between the two nucleases by showing the specific effects of tRNA, ATP, S-adenosyl methionine, and vaccinia antiserum were negative. However, contrary to expectations, while no differential inactivation by heat-
DEOXYRIBONUCLEASES
2
6
IO
FROM
14
2 18 22 Fraction Number
VACCINIA
6
61
VIRUS
IO
14
18
22
FIG. 5. Products of reaction with heated core DNases. Core preparations were kept at 50” for 10,20, and 40 min acc0rdin.g to the protocol of Fig. 4 and used in the reaction for 1 hr at 37” with denatured 3H-labeled B. subtilis DNA. Samples of the reaction mixture were chromatographed as indicated in Fig. 1. Top right: unheated cores; bottom right: lo-min heating; bottom left: 20-min heating; top left: 40-min heating.
ing was observed, there occurred a differential activation of the pH 7.8 enzyme when stripped virus (virus lacking the envelopes) or cores (lacking also lateral bodies) were heated to 50” for 10 min or more (Fig. 4). With stripped virus, the increase of the pH 7.8 nuclease activity was 4-fold and with cores 2.5-fold. It should be mentioned that controlled degradation of stripped virus with trypsin yields cores and enhances both the pH 4.5 and 7.8 activities, although the pH 7.8 enzyme is preferentially stimulated (Pogo and Dales, 1969a; Aubertin and McAuslan, 1972). In the case of cores, analysis of the products of degradation of both enzyme reactions as a function of time of heating to 50” showed that at pH 7.8, after 20 min, there was increased production of oligonucleotides but not of mononucleotides at pH 4.5 (Fig. 5). After 40 min, there was a further increase in oligonucleotides formed. It is clear that heating preferentially stimulated the rate of degradation of DNA by the action of the pH 7.8 nuclease. d. Solubilization
of N&ease
Activities
Differential activation by heat and trypsin strongly suggested that the pH 4.5 and
7.8 activities are present either in different polypeptides or in a single protein with distinctive active centers. Either presumption requires the physical separation of the active enzymes. For this reason, solubilization of the nuclease activities was undertaken. Two methods were used to dissociate cores without inactivating the nucleases: One procedure employed increasing concentrations of NaCl and high ionic strength buffers at neutral pH plus the presence of P-mercaptoethanol; the other used increasing concentrations of urea instead of NaCl with the same additives. Such treatment causes the solubilization of core proteins in varying degrees. Figure 6 shows the amounts of nuclease activity remaining in particulate form after exposure of the cores to increasing concentrations of NaCl or urea. Treatment with 0.5 M salt resulted in solubilization of 80-90% of the acid nuclease but only 20% of the pH 7.8 activity. The latter remained mainly in the particulate form, still part of the DNA-protein complex. The NaCl treatment released 20-30% of the core protein into a soluble form. Extensive exposure of cores to the urea mixture released 50% of the core protein, and both nucleases were
62
POGO
AND
O’SHEA
solubilized (Fig. 6). It is worth mentioning that when 6-8 M urea was added to the nuclease assay mixture containing cores, both activities were stimulated. This effect was less striking when solubilized enzymes were used. The study of the products of degradation by nucleases after salt solubilization (Fig. 7) clearly demonstrated that the activity released by salts in the supernatant was exonucleolytic at pH 4.5 while the one remaining in particulate form was endonucleolytic at pH 7.8. e. Separation of Core Nucleuses lectric Focusing
by Isoe-
The experiments described above, in which enzymatic activities can be differentially solubilized, suggested that they are located in different polypeptide molecules. To substantiate this possibility, the two activities were separated in an electric field by means of isoelectric focusing in either a liquid or a gel milieu. When cores disrupted by sonication were subjected to electrophoresis in a pH gradient from pH 3 to 10, both enzymatic activities were detectable as complexes at various pIs, notably, 8.0, 6.5, and sometimes 5.5 (Fig. 8). However, when enzymes released by NaCl were subjected to electrofocusing, the activities could be clearly separated. The exonuclease banded in the pH gradient at pH 4.5 and the endonuclease at pH 3.5 (Fig. 9). When the products of DNA degradation by the enzymes isolated using electrofocusing were examined, it was found that the enzyme with a p1 of 4.5 yielded mononucleotides and was therefore the exonuclease, while the enzyme with a p1 of 3.5 produced oligonucleotides as expected for an endonuclease. To ascertain the number of proteins involved with the activities, isoelectric focusing in gels was also employed. As illustrated in Fig. 10, separation of the two activities was obtained by electrophoresis in a gradient from pH 3 to 5. The arrows indicate the positions of protein bands observed in companion gels. Two close bands were observed in stained gels that coincided with both activities present at ~15.5. One single band was observed in connec-
01
02
04 M
0.6
0.8
IO
Nacl
FIG. 6. Solubilization of DNases by salt or urea. Core preparations containing 50 pg of protein were resuspended in 0.5 ml of a solution composed of 0.1 M Tris-HCl buffer, pH 7.3, with 0.25% /3-mercaptoethanol and increasing concentrations of NaCl or urea. After 30 min at 4”, the preparations were spun down at 35,000 rpm for 1 hr in an SW 65 rotor. DNase activities were determined in the pellet and supernatant fractions. The figure represents the percentage of nuclease activity that remained in the pellet as a function of the salt or urea concentration used.
tion with each separated enzyme at pIs 4.6 and 3.9.
f.
Polyacrylamide-Gel Electrophoresis the Isolated Enzymes
of
Fractions containing nuclease activities in the liquid isoelectric focusing system were subjected to SDS-gel electrophoresis to determine the molecular weight of the polypeptides in question. In Fig. 11, the stained bands related to the two activities are illustrated. It can be seen that both enzymes occurred in a predominant polypeptide of MW 50,000. The nature of a polypeptide giving a faint band of MW 65,000 which is sometimes present, as
DEOXYRIBONUCLEASES
2
6
IO
14
FROM
IS
22
2
Fraction FIG. 7. Products tant fractions, after (x-x), pH 7.3.
of hydrolysis solubilization
after with
solubilization 0.5 M NaCl,
VACCINIA
6
63
VIRUS
IO
14
IS
22
Number
of DNases with NaCl. Cores and pellet and supernawere assayed as indicated in Fig. 3. (O-O), pH 4.5;
DISCUSSION x--s
p”
.-
77,
56-66
(1977)
Further Characterization BEATRIZ Department
of Cytobiology,
of Deoxyribonucleases G. T. POG02
The Public New
Health York,
Accepted
AND
MICHAEL
Research New York October
Institute 10016
from Vaccinia
Virus’
T. O’SHEA of The
City
of New
York,
Inc.,
27,1976
Further characterization was made of DNases present in vaccinia virus. The nature of the enzymes as they occur within cores and following solubilization was determined. Two activities were identified hydrolyzing single-stranded (ss) DNA but at exclusively a pH optimum of 4.5 or 7.8, respectively. Neither activity has any requirement for ions or cofactors. The pH 7.8 enzyme was activated preferentially by heating cores to 50”. Analysis of the products of hydrolysis by means of DEAE-paper chromatography confirmed that the pH 4.5 activity was an exonuclease and the pH 7.8 enzyme an endonuclease. The exonuclease could act on the 5’-terminus of the DNA. Both nucleases could hydrolyze poly(dT), poly(dA), and poly(dC1 to a varying degree but had no effect on poly(dG). Since the oligonucleotides arising as a product of endonuclease action did not serve as a substrate for the exonuclease, it is concluded that the two enzymes probably do not act in concert. Solubilization of both DNases was achieved by treatment of cores with salts or urea. With 0.5 M NaCl, most of the pH 4.5 activity but only lo-20% of the pH 7.8 was released. The presence of 6-8 M urea caused the solubilization of both enzymes. When in their soluble state, the nucleases could be separated by means of isoelectric focusing in either gel or liquid milieu and retain the exo- or endonucleolytic activities. The pH 4.5 DNase had an isoelectric point or p1 of approximately 4.5, and the pH 7.8 DNase had a p1 of approximately 3.7. Each activity was contained in a single protein. Further analysis of the isolated enzymes, using sodium dodecyl sulfate (SDS)polyacrylsmide-gel electrophoresis, revealed each to be a polypeptide of MW 50,000. Taken together, the evidence indicates that vaccinia cores contain two DNases with indenendent modes of action associated with separate proteins of similar molecular weights but different ~1s.
Pogo and Dales (1969a). Subsequently, other poxviruses were shown to possess similar enzymes, including rabbit pox (Aubertin and McAuslan, 1972), an insect pox (Pago et al., 19711, and Yaba monkey virus (Schwartz and Dales, 1971). The assumption that these are two independent enzyme activities was based on a demonstration of two optimum pHs of approximately 5 and 7.8, differences in substrate affinity (K,,), and the fact that the site of action is either exo- or endonucleolytic. Despite these criteria showing two activities, Rosemond-Hornbeak et al. (1974) could purify only one, the acid deoxyribonuclease activity, from vaccinia cores. Relatively little activity at pH 7.8 was reported. The purified enzyme of RosemondHornbeak et al. (1974) possessed both exo-
INTRODUCTION
Infection of cells by poxvirus elicits the induction of several deoxyribonuclease activities (Hanafusa, 1961; Jungwirth and Joklik, 1965; McAuslan, 1965; McAuslan and Kates, 1966, 1967). At least three activities have been described and differentiated according to substrate specificity, optimum pH for activity, site of action, and time of induction during the virus cycle (McAuslan and Kates, 1966; Pogo and Dales, 196913, 1971). The presence in cores from highly purified preparations of vaccinia virus of two of these nucleases, both specific for singlestranded (ss) DNA, was first reported by 1 This work was supported in part by Health Service Grant AI-12188. 2 Address reprint requests to Dr. Pogo.
Public
56 Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0042-6822
DEOXYRIBONUCLEASES
and endonucleolytic activities and had an estimated molecule weight of 105,000. However, in sodium dodecyl sulfate (SDS) gels the enzyme showed a single polypeptide of MW 50,000, suggesting that the protein is composed of two identical subunits. The discrepancy between previous fmdings and those of Rosemond-Hornbeak et al. (1974) led to the present investigation, intended to establish whether the DNases occur in or&y a single or in two polypeptides of the virus cores. MATERZALS
AND
METHODS
The IHDW strain of vaccinia virus and L, cells were used in accordance with described experimental procedures (Dales, 1963). Methods for the purification of virus particles, preparation of stripped virus and cores, as well as the assays for the nuclease activities have been published (Pago and Dales, 1969a). Solubilization of virion nucleases was obtained by the following procedure: Core preparations containing approximately 50 kg of viral proteins were resuspended in 0.5 ml of 0.1 M Tris-HCl buffer, pH 7.4, with 0.25% P-mercaptoethanol and increasing concentrations of NaCl (0.05 to 1 M) or urea (1 to 8 M). The mixture, kept at 4” for 30 min, was intermittently agitated on a Vortex mixer. The preparations were then spun for 60 min at 35,000 rpm in an SW 65 rotor employing adaptors for 0.5ml nitrocellulose tubes. The pellets obtained were then resuspended in 0.2 ml of 0.01 M Tris-HCl buffer, pH 7.2, and the amount of protein was determined by absorbance at 260 nm. Nuclease activities were measured in both the final pellet and the supernatant fractions. Separation of the virus nucleases by isoelectric focusing was carried out in a liquid system as described by Mandel(1971) or in gels, following the method of Merz et al. (1972). The supernatant samples were extensively dialyzed against 0.01 M TrisHCl buffer, pH 7.2, to remove salts or urea prior to isoelectric focusing. Discontinuous polyacrylamide-gel electrophoresis was performed as described by Obijeski et al. (1973) using as molecular weight standards bovine serum albumin
FROM
VACCINIA
VIRUS
57
(MW 68,000), ovalbumin (MW 45,000), chymotrypsin (MW 25,000>, and cytochrome c (MW 12,000). Products of degradation of the nuclease reactions were characterized as to size by means of paper chromatography (Furlong, 1965). Aliquots of the nuclease reaction mixture were applied to 28cm sheets of Whatman DE81 paper and chromatographed for 4 hr with 1 M NH,HCO,, pH 7.9, and lop3 M EDTA as solvent. The distribution of counts per minute was determined on uniform pieces cut from the chromatogram. Thymidine 5’-triphosphate (TI’P), deoxyadenosine 5’-triphosphate (dATP), and a decanucleotide of ‘M’P were used as markers. Elution of the oligonucleotides from the paper was achieved by incubation with 1 M NH,HCO, for 18 hr at room temperature. Chemicals, isotopes, and enzymes. Thymidine 5’-triphosphate and deoxyadenosine 5’-triphosphate were purchased from Sigma. A decanucleotide of ‘M’P, poly(dC), and poly(dG), as well as labeled 13Hlpoly(dT) (sp act, 12 &i/pm01 of P) and 13Hlpoly(dA> (sp act, 31.4 &i/pmol of P), were obtained from Miles Laboratories. [r32PlATP (sp act, 10.3 Ci/mmol), [814CldATP (sp act, 52.9 mCi/mmol), [53HldTP (sp act, 22.9 Cilmmol), and [83H]dGTP (sp act, 9.4 Ci/mmol) were purchased from New England Nuclear. Calf thymus DNA (from Worthington Co.) with internal and external 5’-32P-labeled termini was prepared following the procedure described by Weiss et al. (1968). For these experiments, pancreatic DNase and alkaline phosphatase from Worthington Biochemical Corp. and polynucleotide kinase from Miles Laboratories were employed. Polynucleotides with 14C-labeled 3’-OH termini were prepared by the action of terminal transferase (from Miles Laboratories) on a [3Hlpoly(dT> or [3Hlpoly(dA) primer. The conditions for this reaction were those described by Chang and Bollum (1971). For the isoelectric focusing experiments, Bio-Lyte ampholytes (Bio-Rad) were employed. 3H-labeled Bacillus subtilis DNA was a generous gift from Dr. David Dubnau of the Department of Microbiology, Public Health Research Institute .
58
POGO
AND
O’SHEA
RESULTS
a. Study of the Products ofDegradation Core Nucleuses
EXONUCLEASE
of
Previous data concerning the presence of exo- and endonucleolytic activities in poxviruses were based on an analysis of the size of degradation products, using acid precipitation, membrane or gel filtration, and sucrose gradients. In the current experiments, ion-exchange paper chromatography, capable of separating polynucleotides according to their chain length, was
Ill
It
I I,.60mm I I I 1
8.000
4,000
h
II
A
2
6
IO
Fractton
II
2
IO
Fraction
14
18
22
FIG. 2. Products of hydrolysis by the exonuclease as a function of reaction time. E. coli exonuclease III, 25 activity units, was incubated with 10 pg of sonicated “H-labeled B. subtilis DNA (sp act, 7000 cpm/pg) at 37” in a l-ml solution containing 0.06 M Tris-HCl, pH 8, 6.6 n&f MgC&, and 1 m&f p-mercaptoethanol. At 15 and 60 min, samples were withdrawn and chromatographed as indicated in Fig. 1.
3H DNA
6
14
Number
IS
22
Number
FIG. 1. Products of hydrolysis by DNase as a function of reaction time. Pancreatic DNase (2130 U/mgl, 30 pg, was incubated at 37” with 10 Fg of 3HIabeledB. subtilis DNA (sp act, 7000 cpm/pgl in 0.5ml solutions containing 0.05 M Tris-HCl, pH 7.8, and 5 mM MgCl,. At 30 and 60 min of incubation, samples were withdrawn and applied to Whatman DE81 paper and chromatographed with 1 M NH,HCO,, pH 7.9, and 10e3 M EDTA for 4 hr. The radioactivity was determined on pieces cut from the chromamgram. The arrows indicate the migration of DNA, a decanucleotide of TMP, and TTP (left to right).
used to analyze the products more directly. Two well-known nucleases were employed for comparative purposes. Labeled DNA was incubated with pancreatic DNase, an endonuclease, or with Escherichiu coli exonuclease III, and the products of hydrolysis were sampled for assay by paper chromatography at intervals during incubation. It is evident in Fig. 1 that, after action of the endonuclease for 30 min, the DNA products had sizes ranging from that of large molecules to 10 nucleotides in length. By 60 min, the only products were short-length polynucleotides and mononucleotides. In contrast, in the case of exonuclease, the size distribution of the products was quite different (Fig. 2); only mononucleotides were observed at 15 or 60 min of incubation.
DEOXYRIBONUCLEASES 1 pti
FROM VACCINIA 1
I
15mln
7 6
10.000
pH4
I
5
400 A
21,500 b 1,800 6:
59
VIRUS
h
30 mln
x
:I, 1I 1
l0,000
d
500. I” \
300
100 7mL
I
I ,x. ,x-i x .
4
8
I I2
I6
J
(/
t
‘r-a 20
Fraction
Number
FIG. 3. Products of hydrolysis by the core-associated DNases as a function of reaction time. Vaccinia cores, 10 pg, were incubated at 37” with 5 pg of denatured 3H-labeledB. subtilis DNA (sp act, 7000 cpm/pg) in 0.2-ml solut.ions containing either 0.05 M Tris-HCl buffer, pH 7.8, or 0.05 M Na-acetate buffer, pH 4.5, both with 0.5% NP40 and 0.25% P-mercaptoethanol. After 15, 30, and 60 min, aliquots were withdrawn and subjected to chromatography as indicated in Fig. 1.
When vaccinia cores were incubated with single-strand-labeled DNA at one or the other of two optimum pHs, the products of hydrolysis accumulating as a function of time were predominantly mono- or dinucleotides in the case of the pH 4.5 reaction, regardless of the time of incubation (Fig. 3); but the reaction at pH 7.8 yielded mostly oligonucleotides, although mono- or dinucleotides were also produced as a function of time (Fig. 2); suggesting processive endonucleolytic activity. These
results clearly indicate that at acid pH the core-associated nuclease activity is exclusively exonucleolytic and at pH 7.8 predominantly endonucleolytic. b. Site of Action
and Substrate
Specificity
DNase-treated calf thymus DNA labeled internally and externally at the 5’-termini with 32P, synthetic homopolydeoxyribonucleotides, and deoxyribopolynucleotides labeled at the 3’-OH ends were used as sub-
60
POGO
AND
strates for the core nucleases. When cores were incubated with appropriately labeled calf thymus ssDNA, it was found that at pH 4.5 there was a release of 32P label, indicating that the exonuclease functions at the Y-terminus. In contrast, there was no release of the 32P label at pH 7.8 (Table 11, indicating that the substrate lacked specific sequences for the endonucleolytic attack. When cores were incubated at pH 4.5 with [3H]poly(dA) as the substrate, in which the 3’-OH end was labeled with 14C, there was no release of the 14C label, indicating that the exonuclease does not attack the 3’OH end (not shown). When homopolydeoxyribonucleotides were used as the substrates, the data summarized in Table 2 showed that the pH 4.5 activity caused hydrolysis of more poly(dT) than poly(dC) and least of poly(dA) but failed to act on poly(dG). A similar order of substrate preference was shown at pH 7.8. To find out whether core nucleases work in a coordinate manner, oligonucleotides, produced by the action of the endonucleolytic cleavage, were used as substrates for the exonuclease. The results of four independent experiments indicated that the exonuclease failed to act on the products of the endonuclease reaction, suggesting that the two activities are not coordinate. This may also imply that the products of endoTABLE SITE
OF ACTION
ON ssDNA
Additions
Buffer, Buffer, Buffer, Buffer,
pH 4.5 pH 4.5, pH 7.8 pH 7.8,
1 BY THE
CORE
DNASES
[32P15’-DNA
+ cores + cores
(cpm)”
(% cpm added)”
9000.0 4850.0 8000.0 8650.0
90.0 48.5 80.0 86.5
o Expressed as acid-precipitable counts per minute. Aliquots of denatured calf thymus DNA carrying $*P-labeled 5’-termini (10,000 cpm) were incubated with 5 pg of cores in a buffered solution containing 0.5% NP40 and 0.25% P-mercaptoethanol. The reaction, allowed to occur for 60 min at 37”, was arrested by the addition of cold 10% trichloroacetic acid and 50 pg of calf thymus DNA as carrier. The precipitates formed were collected on Millipore filters for scintillation counting.
O’SHEA TABLE SUBSTRATE
2
SPECIFICITY
Nucleotide
OF CORE
DNASES
Nucleotide hydrolyzed (nmol/ mg of protein in 60 min)” pH 4.5
pH
7.8
Poly(dA) Poly(dC) Poly(dG)
1200.0 1500.0
650.0 1220.0 -
Poly(dT)
2000.0
1340.0
u [Methyl-“Hlpolydeoxythymidylate (sp act, 12 &ilpmol of P), 12.5 pg; 7 pg of [WHlpolydeoxyadenylic acid (sp act, 31.4 PCilmol of P); 1 A,,, unit of polydeoxycytidylate and 1 A,,, unit of polydeoxyguanylate were incubated for 1 hr at 37” with 5 kg of cores in buffered solutions containing 0.5% NP40 and 0.25% @-mercaptoethanol. The quantity of each polynucleotide hydrolyzed was determined by the amount of acid-soluble counts per minute released or by absorbance at 273 nm in the case of poly(dC) and at 252 nm using poly(dG).
Stripped VIWS ,E 150 E $
CWOC __-’ -1
400
1_____ I:- -----.
.f5 100 I’ h ,’ P ,’ 2 50 I
---*
,’
__--
\
,’
*&.----K-
300
I’
’
,’ 200
5
loa
s Ti0
IO
m
30
40
0
IO
20
3c
40
Minutes ot 50°C
FIG. 4. Effect of elevated temperature on vaccinia DNase activities. Stripped virus or core preparations containing 5 pg of protein in 0.2 ml of 0.01 M Tris-HCl buffer, pH 7.3, and 0.25% P-mercaptoethano1 were heated to 50” for 10, 20, and 40 min, then cooled to 4”. Afterwards, DNase activities were determined in each preparation as described in Materials and Methods.
nucleolytic cleavage have 5’-hydroxyls 3’-phosphates. c. Effect of Elevated &ease Activities
Temperature
and
on Nu-
Attempts to differentiate between the two nucleases by showing the specific effects of tRNA, ATP, S-adenosyl methionine, and vaccinia antiserum were negative. However, contrary to expectations, while no differential inactivation by heat-
DEOXYRIBONUCLEASES
2
6
IO
FROM
14
2 18 22 Fraction Number
VACCINIA
6
61
VIRUS
IO
14
18
22
FIG. 5. Products of reaction with heated core DNases. Core preparations were kept at 50” for 10,20, and 40 min acc0rdin.g to the protocol of Fig. 4 and used in the reaction for 1 hr at 37” with denatured 3H-labeled B. subtilis DNA. Samples of the reaction mixture were chromatographed as indicated in Fig. 1. Top right: unheated cores; bottom right: lo-min heating; bottom left: 20-min heating; top left: 40-min heating.
ing was observed, there occurred a differential activation of the pH 7.8 enzyme when stripped virus (virus lacking the envelopes) or cores (lacking also lateral bodies) were heated to 50” for 10 min or more (Fig. 4). With stripped virus, the increase of the pH 7.8 nuclease activity was 4-fold and with cores 2.5-fold. It should be mentioned that controlled degradation of stripped virus with trypsin yields cores and enhances both the pH 4.5 and 7.8 activities, although the pH 7.8 enzyme is preferentially stimulated (Pogo and Dales, 1969a; Aubertin and McAuslan, 1972). In the case of cores, analysis of the products of degradation of both enzyme reactions as a function of time of heating to 50” showed that at pH 7.8, after 20 min, there was increased production of oligonucleotides but not of mononucleotides at pH 4.5 (Fig. 5). After 40 min, there was a further increase in oligonucleotides formed. It is clear that heating preferentially stimulated the rate of degradation of DNA by the action of the pH 7.8 nuclease. d. Solubilization
of N&ease
Activities
Differential activation by heat and trypsin strongly suggested that the pH 4.5 and
7.8 activities are present either in different polypeptides or in a single protein with distinctive active centers. Either presumption requires the physical separation of the active enzymes. For this reason, solubilization of the nuclease activities was undertaken. Two methods were used to dissociate cores without inactivating the nucleases: One procedure employed increasing concentrations of NaCl and high ionic strength buffers at neutral pH plus the presence of P-mercaptoethanol; the other used increasing concentrations of urea instead of NaCl with the same additives. Such treatment causes the solubilization of core proteins in varying degrees. Figure 6 shows the amounts of nuclease activity remaining in particulate form after exposure of the cores to increasing concentrations of NaCl or urea. Treatment with 0.5 M salt resulted in solubilization of 80-90% of the acid nuclease but only 20% of the pH 7.8 activity. The latter remained mainly in the particulate form, still part of the DNA-protein complex. The NaCl treatment released 20-30% of the core protein into a soluble form. Extensive exposure of cores to the urea mixture released 50% of the core protein, and both nucleases were
62
POGO
AND
O’SHEA
solubilized (Fig. 6). It is worth mentioning that when 6-8 M urea was added to the nuclease assay mixture containing cores, both activities were stimulated. This effect was less striking when solubilized enzymes were used. The study of the products of degradation by nucleases after salt solubilization (Fig. 7) clearly demonstrated that the activity released by salts in the supernatant was exonucleolytic at pH 4.5 while the one remaining in particulate form was endonucleolytic at pH 7.8. e. Separation of Core Nucleuses lectric Focusing
by Isoe-
The experiments described above, in which enzymatic activities can be differentially solubilized, suggested that they are located in different polypeptide molecules. To substantiate this possibility, the two activities were separated in an electric field by means of isoelectric focusing in either a liquid or a gel milieu. When cores disrupted by sonication were subjected to electrophoresis in a pH gradient from pH 3 to 10, both enzymatic activities were detectable as complexes at various pIs, notably, 8.0, 6.5, and sometimes 5.5 (Fig. 8). However, when enzymes released by NaCl were subjected to electrofocusing, the activities could be clearly separated. The exonuclease banded in the pH gradient at pH 4.5 and the endonuclease at pH 3.5 (Fig. 9). When the products of DNA degradation by the enzymes isolated using electrofocusing were examined, it was found that the enzyme with a p1 of 4.5 yielded mononucleotides and was therefore the exonuclease, while the enzyme with a p1 of 3.5 produced oligonucleotides as expected for an endonuclease. To ascertain the number of proteins involved with the activities, isoelectric focusing in gels was also employed. As illustrated in Fig. 10, separation of the two activities was obtained by electrophoresis in a gradient from pH 3 to 5. The arrows indicate the positions of protein bands observed in companion gels. Two close bands were observed in stained gels that coincided with both activities present at ~15.5. One single band was observed in connec-
01
02
04 M
0.6
0.8
IO
Nacl
FIG. 6. Solubilization of DNases by salt or urea. Core preparations containing 50 pg of protein were resuspended in 0.5 ml of a solution composed of 0.1 M Tris-HCl buffer, pH 7.3, with 0.25% /3-mercaptoethanol and increasing concentrations of NaCl or urea. After 30 min at 4”, the preparations were spun down at 35,000 rpm for 1 hr in an SW 65 rotor. DNase activities were determined in the pellet and supernatant fractions. The figure represents the percentage of nuclease activity that remained in the pellet as a function of the salt or urea concentration used.
tion with each separated enzyme at pIs 4.6 and 3.9.
f.
Polyacrylamide-Gel Electrophoresis the Isolated Enzymes
of
Fractions containing nuclease activities in the liquid isoelectric focusing system were subjected to SDS-gel electrophoresis to determine the molecular weight of the polypeptides in question. In Fig. 11, the stained bands related to the two activities are illustrated. It can be seen that both enzymes occurred in a predominant polypeptide of MW 50,000. The nature of a polypeptide giving a faint band of MW 65,000 which is sometimes present, as
DEOXYRIBONUCLEASES
2
6
IO
14
FROM
IS
22
2
Fraction FIG. 7. Products tant fractions, after (x-x), pH 7.3.
of hydrolysis solubilization
after with
solubilization 0.5 M NaCl,
VACCINIA
6
63
VIRUS
IO
14
IS
22
Number
of DNases with NaCl. Cores and pellet and supernawere assayed as indicated in Fig. 3. (O-O), pH 4.5;
DISCUSSION x--s
p”
.-
