Eur. J. Biochem. lY1,263-273 (1990)
(0FEBS 1990
Comparison of exonucleolytic activities of herpes simplex virus type-1 DNA polymerase and DNase Charles W. KNOPF and Klaus WEISSHART Institut fur Virusforschung, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany (Received December 4, 1989/March 7,1990) - EJB 89 1452
The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5’ - 3’exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i. e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5’ - 3‘ direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5’ - 3’-exonucleolytic activities of the DNase and the polymerase-associated 3’ - 5’ exonuclease. The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3’-5’ exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase. Herpes simplex virus (HSV) DNA polymerase is the major essential function of the viral DNA synthesis machinery [I]. The availability of well-characterized temperature-sensitive [l - 31 and drug-resistant 14- 61 DNA polymerase mutants and recent sequencing information [7- 91 render the viral polymerase an attractive model enzyme for studies of eukaryotic DNA polymerases. As a further powerful tool for the biochemical analysis we have recently established polyclonal antibodies directed against selected domains of the polymerase polypeptide. With these monospecific antibodies it was possible to demonstrate [lo], in agreement with the potential coding sequences and the mapping limits for the catalytic domain [9], that HSV type-1 (HSV-1) DNA polymerase of strain Angelotti consists of a single 132 & 5-kDa polypeptide, and that the C-terminal half of the protein is largely responsible for the polymerizing function of the enzyme. HSV-1 DNA polymerase has been shown to be equipped with a deoxyribonuclease activity as initially described by Weissbach and coworkers [l I], that later on was identified as a 3’- 5’ exonuclease [12]. Such a proofreading function seems to belong to the regular outfit of eukaryotic DNA replicases [I3 - 171. From sequencing studies and site-directed mutagenesis [18,19] it is now becoming evident that prokaryotic and eukaryotic DNA polymerases contain a conserved domain for _
_
~
Correspondence to C. W. Knopf, Institut fur Virusforschung, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg 1, Federal Republic of Germany Dedication. This article is dedicated to Professor Dr Arthur Weissbach. Abbreviations: HSV, herpes simplex virus; ts, temperature sensitive; Tris/EDTA, 10 mM Tris/Cl, pH 8.0, 1 mM EDTA. Enzymes. DNA polymerase (EC 2.7.7.7); exodeoxyribonuclease (EC 3.1.11. -); RNA polymerase (EC 2.7.7.6); T4 polynucleotide kinase (EC 2.7.1.78); bacterial alkaline phosphatase (EC 3.1.3.1); ribonuclease A (EC 3.1.27.5); dNTP: DNA deoxynucleotidyl transferase (RNA-directed) (EC 2.7.7.49).
such a proofreading function. Moreover, sequence similarities with the 5‘ - 3‘ exonuclease of Escherichia coli DNA polymerase I have been proposed to occur at the N-terminal region of the human herpes viral DNA polymerases 1191. A main contaminant of HSV DNA polymerase preparations is the virus-induced DNase activity [12]. This nuclease was first characterized as an alkaline nuclease activity 120, 211. HSV-1 DNase was previously shown to be a 85-kDa polypeptide 1221 possessing both 5‘- and 3’-exonuclease activity and also an endonuclease activity 123- 261. The importance of this nuclease, identified as product of gene UL12 [27], for viral replication is still a matter of controversy. Its function is clearly dispensable during transient viral plasmid replication [28] but seems to be essential for replication and growth of the virus in vivo [29]. From an analysis of temperature-sensitive mutants of HSV-2 alkaline DNase [29] it can be inferred that a close functional relationship exists between the viral DNase and DNA polymerase. In order to demonstrate more clearly that the 3’ - 5’ exonuclease is an integral function of the viral DNA polymerase, and further to prove whether an additional 5’ - 3’ exonuclease is associated with this enzyme, which could have escaped detection in the previous analysis [12], we performed a comparative analysis of the nuclease activities associated with the viral DNA polymerase and the alkaline DNase. This study was assisted by our present knowledge of HSV genetics and immunobiochemistry, thus taking advantage of well-characterized temperature-sensitive and drug-resistant polymerase mutants along with the recently established monospecific anti(HSV DNA polymerase) sera. MATERIALS AND METHODS Radiochemicals were purchased from Amersham Buchler (Braunschweig). Nucleotides and synthetic polymers were obtained from Pharmacia LKB (Freiburg) and Boehringer
264 Mannheim. Aphidicolin was from Boehringer Mannheim. The disodium salt of phosphonoacetic acid was obtained from Abbott Laboratories. Enzymes were routinely obtained from Gibco/BRL (Eggenstein), Pharmacia LKB (Freiburg) and Boehringer Mannheim. Growth of cells and virus
glycol, concentrated with the Centricon 30 000 microconcentrator (Amicon Division, W. R. Grace & Co., Danvers) and stored at - 20" C before use. For further purification aliquots of the glycerol-gradientpurified enzyme were loaded on a 0.5-ml phosphocellulose column and washed with column equilibration buffer (20 mM TrisjCI, pH 7.5, 50 mM NaCl, 2 mM EDTA, 2 mM 2-mercaptoethanol). Elution was performed stepwise with the column equilibration buffer containing 35% (by vol.) ethylene glycol and the stated NaCl concentrations.
African green monkey kidney monolayer cells (Rita clone, RC-37; ltaldiagnostic Products, Rome) and baby hamster kindey cells were cultivated in Dulbecco's modified Eagle's medium (Biochrom KG, Berlin) as previously described [30]. Standard assay for HSV D N A polymerase Virus stocks of standard HSV type-I (HSV-1) strain Angelotti Reaction mixtures contained in a total volume of 100 pl: were prepared as described [lo]. HSV-1 strain KOS and the mutants tsC7 (1136) and PAAr-5 [6] were a gift of Dr Priscilla 50 mM Tris/Cl, pH 8.0, 50 pg bovine serum albumin (DNaseSchaffer (Sidney Farber Cancer Institute, Boston), and virus free, Gibco BRL, Eggenstein), 0.5 mM dithiothreitol, 7.5 mM stocks were prepared in BHK-21 cells as previously described MgCl,, 100 mM ammonium sulfate, 0.1 mM each of dATP, M Pcpm/ [31]. Mutant tsC7 exhibited a reversion rate of less than 10W6 dCTP, dGTP, dTTP including one [ E - ~ ~ P I ~ N(800 when assayed at 34" C and 38.5" C. For preparation of infected pmol), 25 pg activated salmon-sperm DNA and enzyme as cells, confluent monolayers were infected at the permissive indicated. After incubation at 37°C for 20 min aliquots of the temperature for a given virus at a multiplicity of ten plaque- reaction mixture were spotted onto Whatman GFjC glass forming units/cell. Cells were collected 18 h after infection by filters and treated to determine acid-insoluble radioactivity as scraping and centrifugation, and the cell pellets were stored described in [12]. at - 70" C before use. Exonuclease assuy Antiseru
Rabbit anti-(HSV-2 DNase) serum raised against purified HSV-2 alkaline DNase was a generous gift of Dr L. M. Banks, University of Leeds. The preparation and characterization of rabbit polyclonal anti-(HSV DNA polymerase) sera were described elsewhere [lo]. Preparation of cell extracts
Dishes (60 mm) with confluently grown BHK-21 cell monolayers were mock and HSV-1 KOS-infected at a multiplicity of infection of five plaque-forming units/cell. At the stated times after infection cells were scraped into 200 pl lysis buffer containing 0.25 M potassium phosphate, pH 7.5,2 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (freshly prepared 100 mM stock solution in 2-propanol) and 0.5% Nonidet P-40 and disrupted by ultrasonication. After centrifugation (10 000 x g, 10 min, 4" C j two parts of the supernatants were mixed with one part ethylene glycol and stored at - 20°C before use.
Exonucleolytic activities were determined with the substrates as indicated below under the conditions of the standard assay for HSV DNA polymerase, except that dNTPs were omitted unless otherwise stated, or of the HSV DNase assay. In brief, the latter reaction mixture contained in a total volume of 100 pl, 50 pg bovine serum albumin, 50 mM Tris/Cl, pH 9, 5 mM MgCl,, 10 mM dithiothreitol, 5 ~g substrate and enzyme as indicated. At the indicated times after incubation at 37°C aliquots were analyzed either by TLC of by homochromatography [32]. RNase-H assuy
Reaction mixtures contained in a total volume of 25 pl: 50 mM TrisjCI, pH 8.0, 1 mM dithiothreitol, 2 mM MnCl,, 1.25 pg p ~ l y ( [ ~ ~ P .] poly(dA) U) (2 x l o 5 cpm/pg) and enzyme as indicated. Reaction mixtures with Moloney murine reverse transcriptase (Stratagene, La Jolla) contained in addition 10 mM MgC12. After incubation at 37" C for 30 min aliquots of the reaction mixtures were analyzed by homochromatography.
Purification of HSV D N A polymerase Polyethylenimine-cellulose thin-layer chromatography The viral enzyme was purified on a small scale from about 10 g frozen HSV-infected cells up to the phosphocellulose Aliquots of reaction mixtures were applied together with chromatography step as previously described [I 21. Aliquots 0.1 pmol marker nucleotide on to polyethyleneimine-cellulose (0.5 - 1 ml) of the HSV DNA polymerase peak fractions were thin-layer sheets (Merck, Darmstadt). For separating pyrimloaded onto glycerol density gradients [polyallomer tubes; idine nucleotides the sheets were developed twice with distilled 15- 30% (by vol.) glycerol in 20 mM Tris/Cl, pH 7.5, 1 mM water up to the origin, followed by 1 M acetic acid to 2 cm 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, above the origin and finally with 0.85 M LiC1. To separate 0.2 M NaCl]. Velocity sedimentation was performed in a purine nucleotides 1 M K H 2 P 0 4 was used as solvent. The Spinco SW 40 rotor at 36000 rpm for 64 h at 2°C. The sedi- nucleotides beeing examined were localized by ultraviolet abmentation standards used were: catalase (244 kDa, 11.3s; sorption as well as by autoradiography and their radioactivity Serva, Heidelberg), lactate dehydrogenase (136 kDa, 6.938; was measured in a liquid scintillation counter. Worthington Biochemical Corp.) and bovine serum albumin (68 kDa, 4.4s; Sigma, Deisenhofen). Peak fractions of HSV Homochromatography DNA polymerase were dialyzed against 50 mM Tris/Cl, To resolve oligomeric degradation products aliquots of pH 7.5, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 50% (by vol.) glycerol or 40% (by vol.) ethylene the reaction mixtures were applied on to polyethyleneimine-
265 cellulose thin-layer sheets. After two washes with water up to the origin, the sheets were developed at 65°C with a neutralized yeast RNA digest containing 3% (mass/vol.) yeast RNA (Boehringer Mannheim) and 7 M urea, as described in [32], and analyzed by autoradiography. Preparation of substrates
Unless otherwise stated, the reaction mixtures were incubated for 30 rnin at 37°C and the substrates were extracted with phenol/chloroform/isoamylalcohol,purified by gel filtration, precipitated with ethanol [43] and resuspended in 10 mM Tris/Cl, pH 8.0, 1 mM EDTA (Tris/EDTA) at a template concentration of 0.5 mg/ml. 3’q3’ PIdNMP-labelled D N A Reaction mixtures contained in a final volume of 100 pl: 1.5 mg/ml activated salmon-sperm DNA prepared as described in [33], 50 pg bovine serum albumin, 50 mM Tris/Cl, pH 7.8, 1 mM dithiothreitol, 5 mM MgC12, 0.1 mM of the stated [a-32P]dNTP(2200 cpm/pmol), 0.2 mM of each of the other three dNTP and 18 units Klenow enzyme (Boehringer Mannheim’).
5‘-3’P-labeled poly ( d T ) and poly ( d T ) . poly ( d A ) Reaction mixtures (50 111) containing 10 mM Tris/Cl, pH 9, 20 pg/ml poly(dT) (160 kDa) and 25 units/ml bacterial alkaline phosphatase (Gibco BRL, Eggenstein) were incubated at 45°C for 30 min. After repeated extraction with phenol/chloroform and ether, the lyophilized poly(dT) was resuspended in 20 p1 reaction mixture containing 50 mM Tris/ C1, pH 7.6, 10 mM MgC12, 5 mM dithiothreitol, 0.1 mM spermidine, 0.1 mM EDTA, 75 pCi [y-32P]ATP (3000 Ci/ mmol) and 20 units T4 polynucleotide kinase (Boehringer Mannheim). To the synthesized 5’-[32P]poly(dT) unlabeled poly(dT) was added to achieve the indicated specific activities. Before use the homopolymer mixture was heated for 5 rnin at 65“C, thereafter rapidly chilled in ice. For preparing 5‘-[32P]poly(dT). poly(dA) three parts (by mass) 5’-[32P]poly(dT)and seven parts (by mass) poly(dA) were heated in Tris/EDTA at 65°C for 5 min, and annealed by slowly cooling to room temperature. ( 3 2 P J d T M P - and ( 3
HldTMP-labeled poly ( d T ) . poly ( d A )
Reaction mixtures contained in a final volume of 100 pl: 250 pg/ml (dT)12- 18 poly(dA) (1 :50, by mass), 50 mM Tris/ C1, pH 7.8, 5 mM MgC12, 1 mM dithiothreitol, 50 pg bovine serum albumin, 0.05 mM each of [u-~’P]~TTP (5000 cpm/ pmol) and dATP, and 30units/ml Klenow enzyme (Boehringer Mannheim). After incubation at 37°C for 5 min, reaction mixtures were phenol/chloroform-extracted, desalted by gel filtration, and the template ethanol-precipitated, washed with 80% ethanol and resuspended in 100 p1 reaction mixture containing 50 mM Tris/Cl, pH 7.8, 5 mM MgC12, 1 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 0.05 mM each of dTTP and dATP, and 60 units/ml Klenow enzyme. After incubation at 37°C for 10 min 30 pCi [3H]dTTP (4400 cpm/pmol) was added, and the incubation was continued for further 10 min. After phenol/chloroform extraction, desalting and ethanol precipitation, the template was resuspended in 100 p1 Tris/EDTA. On average one 32P-labeled sector of about 30 nucleotides was i n ~ o r p o r a t e d / ( d T-) ~1 8~
primer followed by an unlabeled sector of about 60 nucleotides and an 3H-labeled sector of about 60 nucleotides at the 3’ terminus. Poly(dA) . p ~ l y ( [ ~ ~ P ] d T )
Reaction mixtures contained in 100 pl: 50 mM Tris/Cl, pH 7.8, 5 mM MgC12, 1 mM dithiothreitol, 50 pg bovine serum albumin, 0.1 mM [ M - ~ ~ P I ~ T(2000 T P cpm/pmol), 12.5 pg (dT)12-lx . poly(dA) (3:7, by mass) and 5 units Klenow enzyme (Boehringer Mannheim). Poly(dA) . p ~ l y ( ( ~ ’ P ] Uandpoly(dT) ) .p ~ l y ( ( ~ ’ P ] A )
Reaction mixtures contained in 100 pl: 50 mM Tris/Cl, pH 8.0, 5 mM MgC12, 1 mM MnCl,, 1 mM dithiothreitol, 0.1 mM [a-32P]UTP (2000 cpm/pmol), 12.5 pg poly(dA), 5% glycerol and 5 units E. coli RNA polymerase. Poly(dT) . p~ly([~’P]A) was synthesized under identical conditions except that 0.1 mM [LX-~~PIATP and 12.5 pg poly(dT) were used. For the synthesis of poly(dA) . p ~ l y ( [ ~ ~ Pprefer]U) entially labeled at the 5’ or 3’ end, pulse-chase experiments were performed. Reaction mixtures, containing 0.02 mM [a32P]UTP (I0000 cpm/pmol), were incubated for 5 min and chased with 1 mM UTP for 30 rnin in order to incorporate the label at the 5’ terminus. In contrast, reaction mixtures were incubated for 20 min with 0.1 mM cold UTP followed by a 5-min pulse in the presence of [32P]UTP(4000 cpm/pmol) in order to generate a RNA . DNA hybrid preferentially labeled at the 3’ terminus. Polv(A) .p ~ l y ( ( ~ ’ P ] d T )
Reaction mixtures contained in 100 pl: 50 mM Tris/Cl, pH 7.8, 10 mM MgC12, 50 mM KCl, 2 mM dithiothreitol, 50 pg bovine serum albumin, 0.1 mM [a-32P]dTTP(2000 cpm/ pmol) and 30 units Moloney murine reverse transcriptase (Stratagene, La Jolla). RESULTS Further purijkation of HSV D N A polymerase allows the separation of 5’-3’- as well as 3‘ - 5‘-exonucleolytic activities
In order to analyze the exonucleolytic activities associated with HSV DNA polymerase and HSV DNase more closely, the viral DNA polymerase was purified on a small scale following the previously described purification scheme [12]. The phosphocellulose-peak fractions of HSV DNA polymerase of strain KOS were subjected to velocity sedimentation as described in Materials and Methods. As shown in Fig.1 A the KOS DNA polymerase sedimented at 7.5S, corresponding to an apparent molecular mass of 140 kDa [34], and was fairly well separated from HSV DNase (Fig. l A , A) which sedimented at 3.8s. The polymerase-associated 3’ - 5’ exonuclease was the dominant activity when DNase assays were performed under HSV polymerase standard reaction conditions (Fig. l A , A). Further separation of HSV DNase from the glycerolgradient-purified polymerase was attempted by a subsequent phosphocellulose chromatography as shown in Fig. 1B. The majority of DNase activity when monitored under HSV DNase reaction conditions coeluted with the polymerase activity (Fig. l B , A). However, when DNase assays were
266 A
1
2
Table 1. Effect of ionic strength on the hydrolysis o,f single-stranded and double-stranded DNA by the 5’ - 3‘-exonucleolytic activity copuryjing with HSVpolynzerase and DNase Aliquots (5 pl) of HSV polymerase standard reaction mixtures (50 pi) containing 50 pg/ml of the indicated substrates (4 x lo4 cpm pg- I), 1 p l KOS polymerase (glycerol-gradient peak, Fig. 1A) or 1 pl of a 1 : 9 dilution of HSV DNase (glycerol-gradient fractions 16-21, Fig. 1 A) in storage buffer, 0.25 mM each of dATP and dTTP, and the stated concentration of ammonium sulfate, were withdrawn after 0, 5 , 10, 20 and 40 min of incubation at 37 “C and release of 5’[”PIdTMP determined by TLC as described. The presented values were calculated from the initial slopes of the kinetics
3
Ammonium sulfate
40
10
Fraction Number
Fig. 1. Purijication of H S V - I D N A polymerase. (A) Glycerol-gradient velocity sedimentation of HSV-1 DNA polymerase purified from 11.3 g HSV-I-KOS-infected BHK-21 cells as described in Materials and Methods. Sedimentation standards were (1) catalase, (2) lactate dehydrogenase and (3) bovine serum albumin. Aliquots (4 p1) were assayed for HSV DNA polymerase ( 0 )and DNase activity by monitoring the release of [32P]dAMP from 32P-labeled activated DNA (18 pgjml, 6 x lo4 cpm pg- ’) under HSV DNase (A)and polymerase ( A ) reaction conditions as described in the text. (B) Phosphocellulose chromatography of a fraction of the HSV DNA polymerase glycerolgradient peak (A; fractions 10- 13; 0.4 ml). Aliquots (5 111) were assayed for HSV DNA polymerase ( 0 )and for DNase activity with [3ZP]dCMP-labeledactivated DNA (100 pglml, 4 x lo4 cpm pg-’) under HSV DNase conditions ( A ) and with 5’-[3ZP]poly(dT) (50 pg/ ml, 5 x lo4 cpm vg- I ) under polymerase conditions (0)
Release of 5’-[32P]dTMP from __
--
_____-__
5’-[32P]poly(dT) by
5’-[3ZP]p~ly(dT). poly(dA) by
polymerase
DNase
polymerase
DNase
1780 15580 11 840 2190
89 630 959 1402
294 2341 4684 7352
mM
cpm/min
0 25 50 100
890 8310 6470 880
Table 2. Eflect of divalent cations and D N A replication inhibitors on 5’ - 3‘-exonuclease activity Assay conditions were as described in the legend to Table 1. Reaction mixtures contained I00 mM ammonium sulfate, 1 pl of a 1 :9 dilution of HSV DNase (glycerol-gradient peak fraction, Fig. 1 A), and the listcd ingredients. 5’ - 3’-Exonuclease activity was determined as release of 5’-[32P]dTMP from 5’-3ZP-labeled poly(dT) ’ poly(dA) by TLC and calculated from the initial slopes of the kinetics. MalNEt, N-ethylmaleimide; HgHOBzOH, 4-(hydroxymercuri)benzoate Conditions
Control + 5 mM CaCl2 + 0.1 mM ZnS04 + 1 mM ZnS04 + 2.5 mM MalNEt + 0.5 mM HgHOBzOH + 20 pg/ml Phosphonoacetic acid + 5 pgiml Aphidicolin
5’- 3’-Exonuclease activity
cpm/min
%
1352 1510 2407 53 612 0 7340 7363
3 00
21 33 0.7 8 0
performed with 5’-[32P]poly(dT)under the conditions for the HSV DNA polymerase reaction, another major DNase ac100 tivity was identified in the 0.2-M NaCl fractions (Fig. 1B, S), 100 at a salt concentration at which alkaline HSV DNase activity can be eluted from this resin [12]. These results indicated that (a) the phosphocellulose step was sufficient to separate HSV DNA polymerase and HSV DNase, and (b) there was another DNase activity, apparently co-eluting with the HSV DNase that a 5’- 3’-exonucleolytic activity with a strikingly similar but only detectable with a 5‘-labeled substrate, which de- preference for salt was present in both glycerol-gradient engraded DNA in the 5’- 3’ direction. zyme fractions and, to a considerably greater extent, in the HSV DNase peak fractions. The synthetic single-stranded DNA, poly(dT), was preferably hydrolyzed at 25 mM amThe 5’ - 3’-exonucleolytic activity is a property monium sulfate. Degradation of the double-stranded DNA ofthe H S V D N a s r substrate, poly(dT) . poly(dA), increased with increasing ionic To assess the apparent relationship between the novel exo- strength and was enhanced about 20-fold in the presence of nuclease and the HSV DNase, the glycerol-gradient-peak frac- 100 mM ammonium sulfate. This finding was quite surprising tions of HSV DNA polymerase and HSV DNase were exam- since uniformly labeled DNA or DNA preferentially labeled ined for their capabilities to degrade 5’-labeled single- and at the 3’ terminus was degraded tenfold faster by the HSV double-stranded DNA to deoxynucleoside monophosphates DNase in the absence of salt and under standard DNase at different salt concentrations. The possible interference by reaction conditions (Fig. 1). A detailed comparison of assay the polymerase-associated 3’-exonuclease activity at the 3’ conditions revealed that 5’-labeled substrates were in general terminus of the substrates was prevented by the addition of always hydrolyzed more quickly by HSV DNase under HSV deoxynucleotides to the reaction mixtures, i. e. by ongoing polymerase rather than under DNase conditions. Whereas polymerization. As shown in Table 1, the analysis of the re- single-stranded DNA was optimally degraded at 25 mM amleased deoxynucleoside monophosphates by TLC revealed monium sulfate concentration by the HSV DNase, the prefer-
267
B
ori Time
(min)
Fig. 2. Hydrolysis of duplex D N A by H S V D N A polymerase, H S V DNuse and E. coli exonuclease III and analysis of the reaction products. (A) Kinetics of degradation of poly(dT). poly(dA) containing at the 5‘ end of poly(dT) a 32P-labeled and at the 3‘ end an 3H-labeled sector. Reaction mixtures (SO pl) contained 50 pg/ml poly(dT) . poly(dA) (1.2 x lo6 cpm 32P; 5 x l o 5 cpm 3H) and were incubated under HSV DNA polymerase assay conditions in the absence ( 0 , 0) or in the presence of 0.5 mM dATP and dTTP (A,A ) with the following enzymes: (I) 1 p1 KOS polymerase (glycerol-gradient peak, fractions 10-13, Fig. 1 A); (11) 5 pI HSV DNase (phosphocellulose peak, fractions 7-11, Fig. 1 B); (111) 1.25 units E. coli exonuclease 111, (IV) S pl KOS polymerase (phosphocellulose peak, fractions 29 - 35, Fig. Z B). Aliquots ( 5 pl) of the reaction mixtures were withdrawn at the indicated times and the percentage of released 3H- and 32P-labeled dTMP was determined by TLC analysis. The data were corrected for an overlap of 10% cpm 32Pin the 3H channel. (B) Analysis of the degradation products. Aliquots ( 5 pl) of the reaction mixtures (A) after a 30-min incubation at 37°C were analyzed by homochromatography. Lane 1, partial digest of 5’[3ZP]poly(dT)with DNase I. Lane 2, control digestion in the absence of enzyme. Lane 3, HSV DNase reaction mixture (AII). Lane 4,KOS polymerase reaction mixture (AIV). Lane 5, E. coli exonuclease 111 reaction mixture (AIII). Lane 6, 5’-[32P]poly(dT)digested with bacterial alkaline phosphatase
ence for salt varied considerably when double-stranded substrates were used. For example, optimal ammonium sulfate concentrations of 80 mM and 50 mM were determined for the hydrolysis of 5’-[32P]poly(dT). poly(dA) at primer/template ratios of 3 : 7 and of 1: 17 (by mass), respectively. Therefore, in subsequent experiments we performed hydrolysis reactions with 5’-labeled substrates under the reaction conditions described for the HSV DNA polymerase standard assay, i.e. at 100 mM ammonium sulfate. Under these conditions the HSV-DNase-associated 5’ 3’-exonucleolytic activity was not impaired by the common inhibitors of HSV DNA polymerase, phosphonoacetic acid and aphidicolin, at the concentrations stated in Table 2, but the activity was affected by the sulfhydryl-group-blocking agents N-ethylmaleimide and p-hydroxymercuribenzoate, indicating a strong requirement for sulfhydryl groups. In a similar fashion to that previously described for the HSV DNase [23- 251, the novel 5‘ - 3’ exonuclease was inhibited in the presence of 7.5 mM MgC12 by CaZt and Zn2+ ions. According to these data the novel 5’ - 3‘ exonuclease was classified as a property of the HSV DNase. To demonstrate unambiguously the unique exonucleolytic degradation of duplex DNA by HSV DNA polymerase and HSV DNase, a synthetic double-stranded DNA template was constructed which contained a 32P-labeled sector near the 5’ terminus and an 3H-labeled sector at the 3’ terminus of one strand as described in Materials and Methods. With this double-labeled poly(dT) . poly(dA) template it was possible to monitor, at the same time, exonucleolytic degradation events taking place in the 3‘- 5’ and in the 5’- 3’ directions. Fig. 2A shows the results of the hydrolysis of this template by HSV DNA polymerase, HSV DNase and for comparison by E. coli exonuclease I11 under HSV DNA polymerase standard reaction conditions. Strikingly, the phosphocellulose-purified
HSV DNase (Fig. 2A 11) degraded the template exclusively from the 5‘ terminus, i.e. only 5’-[32P]dTMPwas generated within the first 20 min, then 5’-[3H]dTMP was gradually released. In contrast, the HSV-DNA-polymerase-associated exonuclease (Fig. 2A I) hydrolyzed the template in a linear fashion up to 20 min and preferentially from the 3’ terminus by releasing about 4.5 times more 3H-labeled than 32P-labeled dTMP. The E. coli exonuclease I11 (Fig. 2A 111) acted in a similar fashion on this template releasing about four times more [3H]dTMP. The release of both 3H-labeled and 32Plabeled dTMP by the DNA polymerase was completely prevented by the addition of 0.5 mM deoxynucleoside triphosphates, i. e. under conditions of ongoing polymerization (Fig. 2A IV), and demonstrated that most of the hydrolysis exerted by the HSV DNA polymerase took place in the 3‘ - 5’ direction. The analysis of the reaction products by homochromatography [32] revealed that the major 32P-labeled digestion products generated by HSV DNase, HSV DNA polymerase and E. coli exonuclease I11 were deoxynucleoside 5‘monophosphates (Fig. 2 B). Only after longer autoradiographic exposure were oligomeric products identified among the reaction products of the HSV DNase digest, indicating that the endonucleolytic activity [24- 261 of this enzyme was very limited under the conditions for the HSV DNA polymerase reaction. Specificity of the exonucleases associated with the viral D N A polymerase and DNase
The observation that E. coli exonuclease 111 and the nucleases associated with bacterial and eukaryotic DNA polymerases are capable of degrading both strands of DNA . RNA hybrids [43, 441 led us to examine whether the exonucleases
268
:::: B
X
E
::
v
‘
10
5
40 E
O 20
1
C
2
a cu “2
2
3
4
a
b
1 2 2 a 3
1 2 3
5
6
C
1
/ 5
10
20
40
Time (min)
1 2 3
Fig. 3. Churucterizution o f t h e RNuse uctivity ussociuted with HSV DNA polymerase and HSV DNuse. (A) Kinetics of hydrolysis 3‘- and 5‘labeled RNA . DNA hybrids. Reaction mixtures (25 p1) containing 50 pg/ml 3’-[32P]UMP-labeledpoly(U) . poly(dA) (3.9 x 10’ cpm pg- 1) or 5’-[32P]UMP-labeledpoly(U) . poly(dA) (1.3 x lo5 cpm p g - l ; II), and 1 pl HSV DNA polymerase (phosphocellulose fraction; 0 )or 1 p1 HSV DNase (phosphocelluloase fraction; 0)were incubated and processed to determine the released [32P]UMPas described in the legend to Fig. 2A. (B) Degradation of p ~ l y ( [ ~ ~ P.]poly(dA) U) under RNase-H reaction conditions. Reaction mixtures were as described in Materials and Methods and aliquots (4 pl) were analyzed by homochromatography. Lane 1, template control, denatured p01y([~~P]U) . poly(dA) digested with 0.5 pg RNase A. Lane 2, 1 unit E. coli RNase H. Lane 3, 10 units Moloney murine leukemia virus reverse transcriptase. Lane 4, 0.5 pg RNase A. Lane 5 , 2 pI HSV DNA polymerase (phosphocellulose fraction). Lane 6, 2 pl HSV DNase (phosphocellulose fraction). (C) Degradation of RNA . DNA hybrids under HSV DNA polymerase reaction conditions. HSV DNA polymerase reaction mixtures (25 pl) contained alternately 50 pg/ml poly(dA) . p ~ l y ( [ ~ ~ P ](lane U ) I), p01y([~~P]A) . poly(dT) without (lane 2) or with 1 mM each of dATP and dTTP (lane 2a) and denatured p~ly([~’P]A) . poly(dT) (lane 3), and were incubated at 37°C for 30 min with either 1 p1 HSV DNA polymerase (a), 1 p1 HSV DNase (b), or 10 units E. coli exonuclease 111 (c)
Table 3. Specificity of the exonucleolytic activities of HSV DNA polymeruse, HSV DNase and E. coli exonuclease III Reaction conditions were as described in the legend to Fig. 3 C. Release of [32P]NMPfrom thc various RNA . DNA and DNA . DNA hybrids by the enzymes was determined by TLC as described, and is presented as velocities as calculated from the corresponding kinetics. The enzyme concentrations were: 1 pl HSV DNA polymerase (phosphocellulose fraction), 1 1-11 HSV DNase (phosphocellulose fraction) and 10 units E. coli exonuclease I11 (Boehringer Mannheim) Substrate
Release of [32P]NMPby exonuclease 111
polymerase
DNase
pmol . min-
. [enzyme]-
Poly(dA) . p ~ l y ( [ ~ ~ P ] d T0.47 ) Poly(dA) . p ~ l y ( [ ~ ~ P ] U0.31 ) Poly(A) . p~ly([~’P]dT) 6.0 Poly(dT) . p ~ l y ( [ ~ ~ P ] A )0.4 Poly(dT) . p ~ l y ( [ ~ ~ P ] A ) , denatured 0.4
121.8 0.52 11.7 9.7
7.15 0.16 0.2 0.72
7.3
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of the viral DNA polymerase and DNase exhibited similar properties. Various synthetic 32P-labeled RNA . DNA and DNA . DNA hybrids were prepared as described in Materials and Methods, and the degradation of these substrates by the viral enzymes and E. coli exonuclease I11 was analyzed by TLC and homochromatography. As illustrated in Table 3, to
varying degrees each of the enzymes examined was capable of degrading the RNA strands of the synthetic DNA . RNA hybrids. The substrate of choice for the HSV DNase and the exonuclease I11 was the double-stranded DNA hybrid, poly(dA) . poly(dT), which was degraded at least tenfold faster than the RNA . DNA hybrids. In contrast to the latter enzymes, HSV DNA polymerase preferentially degraded the DNA strand of the RNA . DNA hybrids examined, i.e. poly(dT) was hydrolyzed 15 times faster than poly(A) using poly(A) . poly(dT) as substrate. The comparable degradation rates obtained with poly(dT) . p ~ l y ( [ ~ ~ P ]after A ) heat denaturation (Table 3) indicated that all enzymes were also capable of hydrolyzing single-stranded RNA. To determine whether the RNase activity correlated with the nuclease activities associated with viral DNA polymerase and DNase, the degradation of poly(dA) . poly(U), preferentially labeled at the 5’ or 3’ end, by these enzymes was compared. As shown in Fig. 3 A, the 3’-[32P]UMP-labeled poly(dA) . poly(U) was preferably hydrolyzed by the HSV DNA polymerase as was the 5’-labeled DNA . RNA hybrid by the HSV DNase. The major products of the RNA hydrolysis by the viral enzymes were mononucleotides even under RNase-H reaction conditions (Fig. 3 B and C). In addition oligomeric products i. e. mainly di-, tri- and tetra-meric oligoribonucleotides were formed as degradation products of poly(A) . poly(dT) by HSV DNase which revealed that both functions, exonuclease and endonuclease, of this enzyme [24 261 participated in the degradation event. In correlation with the 3’ - 5’-exonuclease function being responsible for the ob-
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20
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Fig. 4. Neutralization of polymerase-associated 3’ -5’ exonuclease and 5’ - 3’ exonuclease by anti- (HSV-2DNaseJ serum. Exonuclease assays were performed with 3’-[32P]dCMP-labeled activated DNA (1.3 x lo5 cpm pg-’) under HSV DNA polymerase reaction conditions (A, B) and with 5’-[32P]poly(dT).poly(dA) (3.3 x lo5 cpm(pmo1 5’(dT) terminus) under HSV DNase reaction conditions (C, D) in the presence of either neutral rabbit serum ( 0 )or anti-(HSV-2 DNase) serum (0).Aliquots of the reaction mixtures containing 5 pl of the glycerol-gradient-purified KOS polymerase (A, C) or HSV DNase (B, D) were analyzed for released [32P]dNMPby TLC as described and the data obtained are presented as percentages of 32P-labeled added DNA
Fig. 5. Induction of HSV DNase in HSV-I KOS infected BHK-21 rells as monitored with 3‘- and 5’-32P-labeled D N A templates. Extracts of mock-( 0 )and HSV-1 KOS( 0)-infected BHK-21 cells were prepared at the indicated times after infection (p.i.) as described in text. Aliquots (1 pl) were assayed with [32P]dCMP-labeled activated DNA (50 pg/ml; 6.5 x lo4 cpm/pg) under HSV DNase standard reaction conditions (I), or with 5’-[3ZP]poly(dT). poly(dA) (50 pg/ml; 1.6 x lo5 cpm/pmol 5’4dT) terminus) under HSV DNA polymerase reaction conditions (11) for 5 min at 37‘C. Released [3ZP]dNMPwas analyzed by TLC as described in the legend to Fig. 4.DNase activity of the mock-infected extracts in experiments (I) and (11) was 1.4% and 0.02%, respectively
served RNase activity, degradation of poly(A) . poly(dT) by the HSV DNA polymerase was completely prevented in the presence of dNTP, i. e. during the ongoing polymerization reaction (Fig. 3 C).
and HSV-1-KOS-infected cells were prepared at different times after infection as described in Materials and Methods and assayed with both, 3’-[32P]dCMP-labeled activated salmon-sperm DNA under optimal HSV DNase conditions and with 5’-32P-labeled poly(dT) . poly(dA) under HSV DNA polymerase conditions. As can be seen in Fig. 5, the amount of released 5’-[32P]dNMPgenerated by the HSV DNase activity of 4-h extracts, for example, was eightfold greater with the 5’-32P-labeled substrate. In addition the background activity was found to be 70-fold lower with the latter template under conditions for the HSV DNA polymerase reaction, and demonstrated that the 5’-32P-labeledtemplate allowed more sensitive detection of HSV DNase. Taking into account that the preferred substrate is single-stranded DNA (Table l), that the specific activity of the 3’-32P-labeledactivated DNA was about twofold higher, and further that the 5’-[32P]poly(dT) consisted of more than 99% unlabeled poly(dT) in order to achieve the substrate concentration of 50 pg/ml, it is obvious that even higher sensitivity of detection of HSV DNase activity cold be realized with a 5’-labeled DNA substrate.
Neutralization of the 5‘ -3‘ exonuclease by anti-(HSV-2 DNase) serum
To prove more directly that HSV DNase contains a cryptic 5’- 3’ exonuclease, we examined whether this enzymatic activity could be neutralized by a monospecific rabbit antiserum directed against the HSV-2 DNase, which was shown to be strongly cross-reactive with the HSV-1-induced enzyme (L. M. Banks and K. Powell, personal communication). For comparison and to confirm the specificity of the antiserum, exonuclease assays were performed with the glycerol-gradientpurified viral enzymes in the presence of rabbit anti-(HSV-2 DNase) serum or control serum, and under the optimal reaction conditions for the respective enzyme. As can be seen from the results of this neutralization experiments presented in Fig. 4, HSV DNase activity was inhibited by 85% and 90% when assayed with a 3’- (Fig. 4B) and 5’-labeled substrate (Fig. 4 D), respectively. The only activity not significantly effected by the anti-(HSV-2 DNase) serum was the polymerase associated 3‘- 5’ exonuclease (Fig. 4A). Improved assay f o r H S V DNase
From the information gained so far it became clear that 5’-labeled DNA templates are the ideal substrates for detecting HSV DNase in cellular extracts. To test this, extracts of mock-
The 3‘ -5‘-exonucleolytic activity is an intrinsic function of the H S V D N A polymerase The following experimental approaches were undertaken to prove that the 3’ - 5’ exonuclease is an intrinsic property of the HSV DNA polymerase polypeptide. Thermolability of both enzymatic activities of mutant tsC7 and wild-type H S V D N A polymerase and oJ’HSV DNase. The HSV-1 KOS polymerase mutant tsC7, whose mutation was mapped to a 1.1-kbp fragment encoding the first 298 amino
270 acid residues [46], induces a DNA polymerase which is thermolabile both in vitro [35] and in vivo [l].We utilized this characteristic to ask whether the polymerase-associated 3' 5' exonuclease is also thermolabile, in order to obtain further evidence for the physical association between exonuclease and polymerase. For this purpose, the tsC7 DNA polymerase was purified from infected BHK-21 cells up to the glycerol-gradient step exactly as described for the corresponding wild-type DNA polymerase of strain KOS in Materials and Methods. Aliquots of the glycerol-gradient-purified enzymes of HSV DNase, tsC7 and KOS DNA polymerase were heat-inacti-
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Fig. 6. Thermolubility qf the exonuclease uctivities associutedwith HSV polymerase and DNase. Nine parts of enzyme in storage buffer were mixed with one part 10 mg/ml bovine serum albumin and incubated at 47' C. (A) At the stated inactivation times aliquots (10 pl) of the preincubation mixtures containing the glycerol-gradient-purified KOS ( 0 , A ) or tsC7 (0, A ) DNA polymerase were assayed both for 3'-exonuclease ( A , A )and polymerase (0,0 )activity under HSV DNA polymerase standard reaction conditions. 100% polymerase activity corresponded to an incorporation of 34.7 pmol and 19.7 pmol dNMP into activated DNA by the KOS and the tsC7 DNA polymerase. respectively. 100% 3'-exonuclease activity corresponded to the release of 21650 and 8530 acid-soluble cpm from 20 Fg/ml [32P]dAMP-labeledactivated DNA (6 x lo4 cpm pg-') by the KOS and tsC7 DNA polymerases, respectively. (B) Aliquots (10 pl) of the identically pretreated HSV DNase glycerol-gradient enzyme were assayed with 60 pg/ml [32P]dAMP-labeledactivated DNA under HSV DNase reaction conditions as described. 100% activity corresponded to 58 700 acid-soluble cpm
vated for different time intervals and then assayed for exonucleolytic and polymerizing activities under the respective optimal reaction conditions. As can be seen from Fig. 6A, the tsC7 DNA polymerase is considerably less stable than the wild-type enzyme. Furthermore, according to the inactivation profiles the polymerase and exonuclease activities of the given DNA polymerase preparations were identically inactivated. The HSV DNase activity (Fig. 6B) was more thermolabile than the exonucleolytic activity associated with the KOS polymerase. Comparison of the drug sensitivity of both enzymatic activities of mutant PAAr-5 and wild-type HSV DNA polymerase. We attempted to gain further information about the close relationship of the polymerizing and exonucleolytic functions of the HSV DNA polymerase by biochemical characterization of the drug-resistant KOS mutant PAA'-5. The PAA'-5 polymerase has recently been shown to carry a mutation at amino acid residue 842 [9] that renders this enzyme resistant to a variety of different antiviral compounds such as phosphonoacetic acid, the triphosphates of acyclovir and dideoxyguanosine and others [36, 371. To study the effect of pyrophosphate analogue upon the enzymatic activities of the PAA'-5 polymerase, the mutant enzyme was purified up to the glycerolgradient step, and its polymerizing and exonucleolytic activities were compared with those of the wild-type enzyme in the presence of phosphonoacetic acid. Fig. 7A shows that, as expected, the polymerizing activity of the PAR-5 enzyme was quite resistant to phosphonoacetic acid, and that in contrast the wild-type polymerase activity was inhibited by 80% with 20 pg/ml phosphonoacetic acid. When the effect of phosphonoacetic acid on the associated exonuclease was examined by measuring the release of 5'-[32P]dGMP from [32P]dGMPlabeled oligo(dG) . poly(dC) in the presence of 20 pg/ml phosphonoacetic acid, we found that the exonucleases exhibited drug sensitivities corresponding to the respective polymerizing activities, as shown in Fig. 7 B. In another set of experiments the effect of the chain terminator ddGTP upon both functions of wild-type and phosphonoacetic-acid-resistant polymerase was compared with that at ongoing polymerization using oligo(dG) . poly(dC) as a template. Incorporation (i. e. polymerization reaction) and excision (i. e. exonucleolytic reaction) of [32P]dGMP by the enzyme was monitored in a time course in the presence of different concentrations of ddGTP by TLC. The results of this analysis (Fig. 8) demonstrated again that exonuclease as well
B
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Fig. 7. &ffects uf phosphonoacetic ucid on polymerizing and exonucleolytic uctivities uf wild-type KOS and PAA'-5 polymeruse. (A) Inhibition of the polymerizing activity of the KOS polymerase ( 0 )and of the PAA'-5 polymerase (A)by phosphonoacetic acid (PAA). 100% activity corresponded to the incorporation of 27 pmol and 7 pmol [32P]dAMPinto activated DNA by 2 pl KOS and PAA'-5 polymerases, respectively, under standard reaction conditions. (B) Inhibition of the 3'-exonuclease activity of KOS (I) and PAAr-5 (11) polymerases by phosphonoacetic acid. Exonuclease activity was monitored as [32P]dGMPreleased from 50 pg/ml 3'-[32P]dGMP-labeled (dG)1Z-18. poly(dC) (5.3 x lo5 cpm pg-'; 1100 cpm/pmol dGMP) by the glycerol-gradient purified KOS (4 pl) and PAAr-5 polymerase (10 pl) in the absence ( 0 ,A ) or in the presence ( 0 ,A) of 20 pg/ml phosphonoacetic acid under HSV D N A polymerase standard reaction conditions as analyzed by TLC
271 A
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Comparison of exonucleolytic activities of herpes simplex virus type-1 DNA polymerase and DNase Charles W. KNOPF and Klaus WEISSHART Institut fur Virusforschung, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany (Received December 4, 1989/March 7,1990) - EJB 89 1452
The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5’ - 3’exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i. e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5’ - 3‘ direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5’ - 3’-exonucleolytic activities of the DNase and the polymerase-associated 3’ - 5’ exonuclease. The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3’-5’ exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase. Herpes simplex virus (HSV) DNA polymerase is the major essential function of the viral DNA synthesis machinery [I]. The availability of well-characterized temperature-sensitive [l - 31 and drug-resistant 14- 61 DNA polymerase mutants and recent sequencing information [7- 91 render the viral polymerase an attractive model enzyme for studies of eukaryotic DNA polymerases. As a further powerful tool for the biochemical analysis we have recently established polyclonal antibodies directed against selected domains of the polymerase polypeptide. With these monospecific antibodies it was possible to demonstrate [lo], in agreement with the potential coding sequences and the mapping limits for the catalytic domain [9], that HSV type-1 (HSV-1) DNA polymerase of strain Angelotti consists of a single 132 & 5-kDa polypeptide, and that the C-terminal half of the protein is largely responsible for the polymerizing function of the enzyme. HSV-1 DNA polymerase has been shown to be equipped with a deoxyribonuclease activity as initially described by Weissbach and coworkers [l I], that later on was identified as a 3’- 5’ exonuclease [12]. Such a proofreading function seems to belong to the regular outfit of eukaryotic DNA replicases [I3 - 171. From sequencing studies and site-directed mutagenesis [18,19] it is now becoming evident that prokaryotic and eukaryotic DNA polymerases contain a conserved domain for _
_
~
Correspondence to C. W. Knopf, Institut fur Virusforschung, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg 1, Federal Republic of Germany Dedication. This article is dedicated to Professor Dr Arthur Weissbach. Abbreviations: HSV, herpes simplex virus; ts, temperature sensitive; Tris/EDTA, 10 mM Tris/Cl, pH 8.0, 1 mM EDTA. Enzymes. DNA polymerase (EC 2.7.7.7); exodeoxyribonuclease (EC 3.1.11. -); RNA polymerase (EC 2.7.7.6); T4 polynucleotide kinase (EC 2.7.1.78); bacterial alkaline phosphatase (EC 3.1.3.1); ribonuclease A (EC 3.1.27.5); dNTP: DNA deoxynucleotidyl transferase (RNA-directed) (EC 2.7.7.49).
such a proofreading function. Moreover, sequence similarities with the 5‘ - 3‘ exonuclease of Escherichia coli DNA polymerase I have been proposed to occur at the N-terminal region of the human herpes viral DNA polymerases 1191. A main contaminant of HSV DNA polymerase preparations is the virus-induced DNase activity [12]. This nuclease was first characterized as an alkaline nuclease activity 120, 211. HSV-1 DNase was previously shown to be a 85-kDa polypeptide 1221 possessing both 5‘- and 3’-exonuclease activity and also an endonuclease activity 123- 261. The importance of this nuclease, identified as product of gene UL12 [27], for viral replication is still a matter of controversy. Its function is clearly dispensable during transient viral plasmid replication [28] but seems to be essential for replication and growth of the virus in vivo [29]. From an analysis of temperature-sensitive mutants of HSV-2 alkaline DNase [29] it can be inferred that a close functional relationship exists between the viral DNase and DNA polymerase. In order to demonstrate more clearly that the 3’ - 5’ exonuclease is an integral function of the viral DNA polymerase, and further to prove whether an additional 5’ - 3’ exonuclease is associated with this enzyme, which could have escaped detection in the previous analysis [12], we performed a comparative analysis of the nuclease activities associated with the viral DNA polymerase and the alkaline DNase. This study was assisted by our present knowledge of HSV genetics and immunobiochemistry, thus taking advantage of well-characterized temperature-sensitive and drug-resistant polymerase mutants along with the recently established monospecific anti(HSV DNA polymerase) sera. MATERIALS AND METHODS Radiochemicals were purchased from Amersham Buchler (Braunschweig). Nucleotides and synthetic polymers were obtained from Pharmacia LKB (Freiburg) and Boehringer
264 Mannheim. Aphidicolin was from Boehringer Mannheim. The disodium salt of phosphonoacetic acid was obtained from Abbott Laboratories. Enzymes were routinely obtained from Gibco/BRL (Eggenstein), Pharmacia LKB (Freiburg) and Boehringer Mannheim. Growth of cells and virus
glycol, concentrated with the Centricon 30 000 microconcentrator (Amicon Division, W. R. Grace & Co., Danvers) and stored at - 20" C before use. For further purification aliquots of the glycerol-gradientpurified enzyme were loaded on a 0.5-ml phosphocellulose column and washed with column equilibration buffer (20 mM TrisjCI, pH 7.5, 50 mM NaCl, 2 mM EDTA, 2 mM 2-mercaptoethanol). Elution was performed stepwise with the column equilibration buffer containing 35% (by vol.) ethylene glycol and the stated NaCl concentrations.
African green monkey kidney monolayer cells (Rita clone, RC-37; ltaldiagnostic Products, Rome) and baby hamster kindey cells were cultivated in Dulbecco's modified Eagle's medium (Biochrom KG, Berlin) as previously described [30]. Standard assay for HSV D N A polymerase Virus stocks of standard HSV type-I (HSV-1) strain Angelotti Reaction mixtures contained in a total volume of 100 pl: were prepared as described [lo]. HSV-1 strain KOS and the mutants tsC7 (1136) and PAAr-5 [6] were a gift of Dr Priscilla 50 mM Tris/Cl, pH 8.0, 50 pg bovine serum albumin (DNaseSchaffer (Sidney Farber Cancer Institute, Boston), and virus free, Gibco BRL, Eggenstein), 0.5 mM dithiothreitol, 7.5 mM stocks were prepared in BHK-21 cells as previously described MgCl,, 100 mM ammonium sulfate, 0.1 mM each of dATP, M Pcpm/ [31]. Mutant tsC7 exhibited a reversion rate of less than 10W6 dCTP, dGTP, dTTP including one [ E - ~ ~ P I ~ N(800 when assayed at 34" C and 38.5" C. For preparation of infected pmol), 25 pg activated salmon-sperm DNA and enzyme as cells, confluent monolayers were infected at the permissive indicated. After incubation at 37°C for 20 min aliquots of the temperature for a given virus at a multiplicity of ten plaque- reaction mixture were spotted onto Whatman GFjC glass forming units/cell. Cells were collected 18 h after infection by filters and treated to determine acid-insoluble radioactivity as scraping and centrifugation, and the cell pellets were stored described in [12]. at - 70" C before use. Exonuclease assuy Antiseru
Rabbit anti-(HSV-2 DNase) serum raised against purified HSV-2 alkaline DNase was a generous gift of Dr L. M. Banks, University of Leeds. The preparation and characterization of rabbit polyclonal anti-(HSV DNA polymerase) sera were described elsewhere [lo]. Preparation of cell extracts
Dishes (60 mm) with confluently grown BHK-21 cell monolayers were mock and HSV-1 KOS-infected at a multiplicity of infection of five plaque-forming units/cell. At the stated times after infection cells were scraped into 200 pl lysis buffer containing 0.25 M potassium phosphate, pH 7.5,2 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (freshly prepared 100 mM stock solution in 2-propanol) and 0.5% Nonidet P-40 and disrupted by ultrasonication. After centrifugation (10 000 x g, 10 min, 4" C j two parts of the supernatants were mixed with one part ethylene glycol and stored at - 20°C before use.
Exonucleolytic activities were determined with the substrates as indicated below under the conditions of the standard assay for HSV DNA polymerase, except that dNTPs were omitted unless otherwise stated, or of the HSV DNase assay. In brief, the latter reaction mixture contained in a total volume of 100 pl, 50 pg bovine serum albumin, 50 mM Tris/Cl, pH 9, 5 mM MgCl,, 10 mM dithiothreitol, 5 ~g substrate and enzyme as indicated. At the indicated times after incubation at 37°C aliquots were analyzed either by TLC of by homochromatography [32]. RNase-H assuy
Reaction mixtures contained in a total volume of 25 pl: 50 mM TrisjCI, pH 8.0, 1 mM dithiothreitol, 2 mM MnCl,, 1.25 pg p ~ l y ( [ ~ ~ P .] poly(dA) U) (2 x l o 5 cpm/pg) and enzyme as indicated. Reaction mixtures with Moloney murine reverse transcriptase (Stratagene, La Jolla) contained in addition 10 mM MgC12. After incubation at 37" C for 30 min aliquots of the reaction mixtures were analyzed by homochromatography.
Purification of HSV D N A polymerase Polyethylenimine-cellulose thin-layer chromatography The viral enzyme was purified on a small scale from about 10 g frozen HSV-infected cells up to the phosphocellulose Aliquots of reaction mixtures were applied together with chromatography step as previously described [I 21. Aliquots 0.1 pmol marker nucleotide on to polyethyleneimine-cellulose (0.5 - 1 ml) of the HSV DNA polymerase peak fractions were thin-layer sheets (Merck, Darmstadt). For separating pyrimloaded onto glycerol density gradients [polyallomer tubes; idine nucleotides the sheets were developed twice with distilled 15- 30% (by vol.) glycerol in 20 mM Tris/Cl, pH 7.5, 1 mM water up to the origin, followed by 1 M acetic acid to 2 cm 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, above the origin and finally with 0.85 M LiC1. To separate 0.2 M NaCl]. Velocity sedimentation was performed in a purine nucleotides 1 M K H 2 P 0 4 was used as solvent. The Spinco SW 40 rotor at 36000 rpm for 64 h at 2°C. The sedi- nucleotides beeing examined were localized by ultraviolet abmentation standards used were: catalase (244 kDa, 11.3s; sorption as well as by autoradiography and their radioactivity Serva, Heidelberg), lactate dehydrogenase (136 kDa, 6.938; was measured in a liquid scintillation counter. Worthington Biochemical Corp.) and bovine serum albumin (68 kDa, 4.4s; Sigma, Deisenhofen). Peak fractions of HSV Homochromatography DNA polymerase were dialyzed against 50 mM Tris/Cl, To resolve oligomeric degradation products aliquots of pH 7.5, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 50% (by vol.) glycerol or 40% (by vol.) ethylene the reaction mixtures were applied on to polyethyleneimine-
265 cellulose thin-layer sheets. After two washes with water up to the origin, the sheets were developed at 65°C with a neutralized yeast RNA digest containing 3% (mass/vol.) yeast RNA (Boehringer Mannheim) and 7 M urea, as described in [32], and analyzed by autoradiography. Preparation of substrates
Unless otherwise stated, the reaction mixtures were incubated for 30 rnin at 37°C and the substrates were extracted with phenol/chloroform/isoamylalcohol,purified by gel filtration, precipitated with ethanol [43] and resuspended in 10 mM Tris/Cl, pH 8.0, 1 mM EDTA (Tris/EDTA) at a template concentration of 0.5 mg/ml. 3’q3’ PIdNMP-labelled D N A Reaction mixtures contained in a final volume of 100 pl: 1.5 mg/ml activated salmon-sperm DNA prepared as described in [33], 50 pg bovine serum albumin, 50 mM Tris/Cl, pH 7.8, 1 mM dithiothreitol, 5 mM MgC12, 0.1 mM of the stated [a-32P]dNTP(2200 cpm/pmol), 0.2 mM of each of the other three dNTP and 18 units Klenow enzyme (Boehringer Mannheim’).
5‘-3’P-labeled poly ( d T ) and poly ( d T ) . poly ( d A ) Reaction mixtures (50 111) containing 10 mM Tris/Cl, pH 9, 20 pg/ml poly(dT) (160 kDa) and 25 units/ml bacterial alkaline phosphatase (Gibco BRL, Eggenstein) were incubated at 45°C for 30 min. After repeated extraction with phenol/chloroform and ether, the lyophilized poly(dT) was resuspended in 20 p1 reaction mixture containing 50 mM Tris/ C1, pH 7.6, 10 mM MgC12, 5 mM dithiothreitol, 0.1 mM spermidine, 0.1 mM EDTA, 75 pCi [y-32P]ATP (3000 Ci/ mmol) and 20 units T4 polynucleotide kinase (Boehringer Mannheim). To the synthesized 5’-[32P]poly(dT) unlabeled poly(dT) was added to achieve the indicated specific activities. Before use the homopolymer mixture was heated for 5 rnin at 65“C, thereafter rapidly chilled in ice. For preparing 5‘-[32P]poly(dT). poly(dA) three parts (by mass) 5’-[32P]poly(dT)and seven parts (by mass) poly(dA) were heated in Tris/EDTA at 65°C for 5 min, and annealed by slowly cooling to room temperature. ( 3 2 P J d T M P - and ( 3
HldTMP-labeled poly ( d T ) . poly ( d A )
Reaction mixtures contained in a final volume of 100 pl: 250 pg/ml (dT)12- 18 poly(dA) (1 :50, by mass), 50 mM Tris/ C1, pH 7.8, 5 mM MgC12, 1 mM dithiothreitol, 50 pg bovine serum albumin, 0.05 mM each of [u-~’P]~TTP (5000 cpm/ pmol) and dATP, and 30units/ml Klenow enzyme (Boehringer Mannheim). After incubation at 37°C for 5 min, reaction mixtures were phenol/chloroform-extracted, desalted by gel filtration, and the template ethanol-precipitated, washed with 80% ethanol and resuspended in 100 p1 reaction mixture containing 50 mM Tris/Cl, pH 7.8, 5 mM MgC12, 1 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 0.05 mM each of dTTP and dATP, and 60 units/ml Klenow enzyme. After incubation at 37°C for 10 min 30 pCi [3H]dTTP (4400 cpm/pmol) was added, and the incubation was continued for further 10 min. After phenol/chloroform extraction, desalting and ethanol precipitation, the template was resuspended in 100 p1 Tris/EDTA. On average one 32P-labeled sector of about 30 nucleotides was i n ~ o r p o r a t e d / ( d T-) ~1 8~
primer followed by an unlabeled sector of about 60 nucleotides and an 3H-labeled sector of about 60 nucleotides at the 3’ terminus. Poly(dA) . p ~ l y ( [ ~ ~ P ] d T )
Reaction mixtures contained in 100 pl: 50 mM Tris/Cl, pH 7.8, 5 mM MgC12, 1 mM dithiothreitol, 50 pg bovine serum albumin, 0.1 mM [ M - ~ ~ P I ~ T(2000 T P cpm/pmol), 12.5 pg (dT)12-lx . poly(dA) (3:7, by mass) and 5 units Klenow enzyme (Boehringer Mannheim). Poly(dA) . p ~ l y ( ( ~ ’ P ] Uandpoly(dT) ) .p ~ l y ( ( ~ ’ P ] A )
Reaction mixtures contained in 100 pl: 50 mM Tris/Cl, pH 8.0, 5 mM MgC12, 1 mM MnCl,, 1 mM dithiothreitol, 0.1 mM [a-32P]UTP (2000 cpm/pmol), 12.5 pg poly(dA), 5% glycerol and 5 units E. coli RNA polymerase. Poly(dT) . p~ly([~’P]A) was synthesized under identical conditions except that 0.1 mM [LX-~~PIATP and 12.5 pg poly(dT) were used. For the synthesis of poly(dA) . p ~ l y ( [ ~ ~ Pprefer]U) entially labeled at the 5’ or 3’ end, pulse-chase experiments were performed. Reaction mixtures, containing 0.02 mM [a32P]UTP (I0000 cpm/pmol), were incubated for 5 min and chased with 1 mM UTP for 30 rnin in order to incorporate the label at the 5’ terminus. In contrast, reaction mixtures were incubated for 20 min with 0.1 mM cold UTP followed by a 5-min pulse in the presence of [32P]UTP(4000 cpm/pmol) in order to generate a RNA . DNA hybrid preferentially labeled at the 3’ terminus. Polv(A) .p ~ l y ( ( ~ ’ P ] d T )
Reaction mixtures contained in 100 pl: 50 mM Tris/Cl, pH 7.8, 10 mM MgC12, 50 mM KCl, 2 mM dithiothreitol, 50 pg bovine serum albumin, 0.1 mM [a-32P]dTTP(2000 cpm/ pmol) and 30 units Moloney murine reverse transcriptase (Stratagene, La Jolla). RESULTS Further purijkation of HSV D N A polymerase allows the separation of 5’-3’- as well as 3‘ - 5‘-exonucleolytic activities
In order to analyze the exonucleolytic activities associated with HSV DNA polymerase and HSV DNase more closely, the viral DNA polymerase was purified on a small scale following the previously described purification scheme [12]. The phosphocellulose-peak fractions of HSV DNA polymerase of strain KOS were subjected to velocity sedimentation as described in Materials and Methods. As shown in Fig.1 A the KOS DNA polymerase sedimented at 7.5S, corresponding to an apparent molecular mass of 140 kDa [34], and was fairly well separated from HSV DNase (Fig. l A , A) which sedimented at 3.8s. The polymerase-associated 3’ - 5’ exonuclease was the dominant activity when DNase assays were performed under HSV polymerase standard reaction conditions (Fig. l A , A). Further separation of HSV DNase from the glycerolgradient-purified polymerase was attempted by a subsequent phosphocellulose chromatography as shown in Fig. 1B. The majority of DNase activity when monitored under HSV DNase reaction conditions coeluted with the polymerase activity (Fig. l B , A). However, when DNase assays were
266 A
1
2
Table 1. Effect of ionic strength on the hydrolysis o,f single-stranded and double-stranded DNA by the 5’ - 3‘-exonucleolytic activity copuryjing with HSVpolynzerase and DNase Aliquots (5 pl) of HSV polymerase standard reaction mixtures (50 pi) containing 50 pg/ml of the indicated substrates (4 x lo4 cpm pg- I), 1 p l KOS polymerase (glycerol-gradient peak, Fig. 1A) or 1 pl of a 1 : 9 dilution of HSV DNase (glycerol-gradient fractions 16-21, Fig. 1 A) in storage buffer, 0.25 mM each of dATP and dTTP, and the stated concentration of ammonium sulfate, were withdrawn after 0, 5 , 10, 20 and 40 min of incubation at 37 “C and release of 5’[”PIdTMP determined by TLC as described. The presented values were calculated from the initial slopes of the kinetics
3
Ammonium sulfate
40
10
Fraction Number
Fig. 1. Purijication of H S V - I D N A polymerase. (A) Glycerol-gradient velocity sedimentation of HSV-1 DNA polymerase purified from 11.3 g HSV-I-KOS-infected BHK-21 cells as described in Materials and Methods. Sedimentation standards were (1) catalase, (2) lactate dehydrogenase and (3) bovine serum albumin. Aliquots (4 p1) were assayed for HSV DNA polymerase ( 0 )and DNase activity by monitoring the release of [32P]dAMP from 32P-labeled activated DNA (18 pgjml, 6 x lo4 cpm pg- ’) under HSV DNase (A)and polymerase ( A ) reaction conditions as described in the text. (B) Phosphocellulose chromatography of a fraction of the HSV DNA polymerase glycerolgradient peak (A; fractions 10- 13; 0.4 ml). Aliquots (5 111) were assayed for HSV DNA polymerase ( 0 )and for DNase activity with [3ZP]dCMP-labeledactivated DNA (100 pglml, 4 x lo4 cpm pg-’) under HSV DNase conditions ( A ) and with 5’-[3ZP]poly(dT) (50 pg/ ml, 5 x lo4 cpm vg- I ) under polymerase conditions (0)
Release of 5’-[32P]dTMP from __
--
_____-__
5’-[32P]poly(dT) by
5’-[3ZP]p~ly(dT). poly(dA) by
polymerase
DNase
polymerase
DNase
1780 15580 11 840 2190
89 630 959 1402
294 2341 4684 7352
mM
cpm/min
0 25 50 100
890 8310 6470 880
Table 2. Eflect of divalent cations and D N A replication inhibitors on 5’ - 3‘-exonuclease activity Assay conditions were as described in the legend to Table 1. Reaction mixtures contained I00 mM ammonium sulfate, 1 pl of a 1 :9 dilution of HSV DNase (glycerol-gradient peak fraction, Fig. 1 A), and the listcd ingredients. 5’ - 3’-Exonuclease activity was determined as release of 5’-[32P]dTMP from 5’-3ZP-labeled poly(dT) ’ poly(dA) by TLC and calculated from the initial slopes of the kinetics. MalNEt, N-ethylmaleimide; HgHOBzOH, 4-(hydroxymercuri)benzoate Conditions
Control + 5 mM CaCl2 + 0.1 mM ZnS04 + 1 mM ZnS04 + 2.5 mM MalNEt + 0.5 mM HgHOBzOH + 20 pg/ml Phosphonoacetic acid + 5 pgiml Aphidicolin
5’- 3’-Exonuclease activity
cpm/min
%
1352 1510 2407 53 612 0 7340 7363
3 00
21 33 0.7 8 0
performed with 5’-[32P]poly(dT)under the conditions for the HSV DNA polymerase reaction, another major DNase ac100 tivity was identified in the 0.2-M NaCl fractions (Fig. 1B, S), 100 at a salt concentration at which alkaline HSV DNase activity can be eluted from this resin [12]. These results indicated that (a) the phosphocellulose step was sufficient to separate HSV DNA polymerase and HSV DNase, and (b) there was another DNase activity, apparently co-eluting with the HSV DNase that a 5’- 3’-exonucleolytic activity with a strikingly similar but only detectable with a 5‘-labeled substrate, which de- preference for salt was present in both glycerol-gradient engraded DNA in the 5’- 3’ direction. zyme fractions and, to a considerably greater extent, in the HSV DNase peak fractions. The synthetic single-stranded DNA, poly(dT), was preferably hydrolyzed at 25 mM amThe 5’ - 3’-exonucleolytic activity is a property monium sulfate. Degradation of the double-stranded DNA ofthe H S V D N a s r substrate, poly(dT) . poly(dA), increased with increasing ionic To assess the apparent relationship between the novel exo- strength and was enhanced about 20-fold in the presence of nuclease and the HSV DNase, the glycerol-gradient-peak frac- 100 mM ammonium sulfate. This finding was quite surprising tions of HSV DNA polymerase and HSV DNase were exam- since uniformly labeled DNA or DNA preferentially labeled ined for their capabilities to degrade 5’-labeled single- and at the 3’ terminus was degraded tenfold faster by the HSV double-stranded DNA to deoxynucleoside monophosphates DNase in the absence of salt and under standard DNase at different salt concentrations. The possible interference by reaction conditions (Fig. 1). A detailed comparison of assay the polymerase-associated 3’-exonuclease activity at the 3’ conditions revealed that 5’-labeled substrates were in general terminus of the substrates was prevented by the addition of always hydrolyzed more quickly by HSV DNase under HSV deoxynucleotides to the reaction mixtures, i. e. by ongoing polymerase rather than under DNase conditions. Whereas polymerization. As shown in Table 1, the analysis of the re- single-stranded DNA was optimally degraded at 25 mM amleased deoxynucleoside monophosphates by TLC revealed monium sulfate concentration by the HSV DNase, the prefer-
267
B
ori Time
(min)
Fig. 2. Hydrolysis of duplex D N A by H S V D N A polymerase, H S V DNuse and E. coli exonuclease III and analysis of the reaction products. (A) Kinetics of degradation of poly(dT). poly(dA) containing at the 5‘ end of poly(dT) a 32P-labeled and at the 3‘ end an 3H-labeled sector. Reaction mixtures (SO pl) contained 50 pg/ml poly(dT) . poly(dA) (1.2 x lo6 cpm 32P; 5 x l o 5 cpm 3H) and were incubated under HSV DNA polymerase assay conditions in the absence ( 0 , 0) or in the presence of 0.5 mM dATP and dTTP (A,A ) with the following enzymes: (I) 1 p1 KOS polymerase (glycerol-gradient peak, fractions 10-13, Fig. 1 A); (11) 5 pI HSV DNase (phosphocellulose peak, fractions 7-11, Fig. 1 B); (111) 1.25 units E. coli exonuclease 111, (IV) S pl KOS polymerase (phosphocellulose peak, fractions 29 - 35, Fig. Z B). Aliquots ( 5 pl) of the reaction mixtures were withdrawn at the indicated times and the percentage of released 3H- and 32P-labeled dTMP was determined by TLC analysis. The data were corrected for an overlap of 10% cpm 32Pin the 3H channel. (B) Analysis of the degradation products. Aliquots ( 5 pl) of the reaction mixtures (A) after a 30-min incubation at 37°C were analyzed by homochromatography. Lane 1, partial digest of 5’[3ZP]poly(dT)with DNase I. Lane 2, control digestion in the absence of enzyme. Lane 3, HSV DNase reaction mixture (AII). Lane 4,KOS polymerase reaction mixture (AIV). Lane 5, E. coli exonuclease 111 reaction mixture (AIII). Lane 6, 5’-[32P]poly(dT)digested with bacterial alkaline phosphatase
ence for salt varied considerably when double-stranded substrates were used. For example, optimal ammonium sulfate concentrations of 80 mM and 50 mM were determined for the hydrolysis of 5’-[32P]poly(dT). poly(dA) at primer/template ratios of 3 : 7 and of 1: 17 (by mass), respectively. Therefore, in subsequent experiments we performed hydrolysis reactions with 5’-labeled substrates under the reaction conditions described for the HSV DNA polymerase standard assay, i.e. at 100 mM ammonium sulfate. Under these conditions the HSV-DNase-associated 5’ 3’-exonucleolytic activity was not impaired by the common inhibitors of HSV DNA polymerase, phosphonoacetic acid and aphidicolin, at the concentrations stated in Table 2, but the activity was affected by the sulfhydryl-group-blocking agents N-ethylmaleimide and p-hydroxymercuribenzoate, indicating a strong requirement for sulfhydryl groups. In a similar fashion to that previously described for the HSV DNase [23- 251, the novel 5‘ - 3’ exonuclease was inhibited in the presence of 7.5 mM MgC12 by CaZt and Zn2+ ions. According to these data the novel 5’ - 3‘ exonuclease was classified as a property of the HSV DNase. To demonstrate unambiguously the unique exonucleolytic degradation of duplex DNA by HSV DNA polymerase and HSV DNase, a synthetic double-stranded DNA template was constructed which contained a 32P-labeled sector near the 5’ terminus and an 3H-labeled sector at the 3’ terminus of one strand as described in Materials and Methods. With this double-labeled poly(dT) . poly(dA) template it was possible to monitor, at the same time, exonucleolytic degradation events taking place in the 3‘- 5’ and in the 5’- 3’ directions. Fig. 2A shows the results of the hydrolysis of this template by HSV DNA polymerase, HSV DNase and for comparison by E. coli exonuclease I11 under HSV DNA polymerase standard reaction conditions. Strikingly, the phosphocellulose-purified
HSV DNase (Fig. 2A 11) degraded the template exclusively from the 5‘ terminus, i.e. only 5’-[32P]dTMPwas generated within the first 20 min, then 5’-[3H]dTMP was gradually released. In contrast, the HSV-DNA-polymerase-associated exonuclease (Fig. 2A I) hydrolyzed the template in a linear fashion up to 20 min and preferentially from the 3’ terminus by releasing about 4.5 times more 3H-labeled than 32P-labeled dTMP. The E. coli exonuclease I11 (Fig. 2A 111) acted in a similar fashion on this template releasing about four times more [3H]dTMP. The release of both 3H-labeled and 32Plabeled dTMP by the DNA polymerase was completely prevented by the addition of 0.5 mM deoxynucleoside triphosphates, i. e. under conditions of ongoing polymerization (Fig. 2A IV), and demonstrated that most of the hydrolysis exerted by the HSV DNA polymerase took place in the 3‘ - 5’ direction. The analysis of the reaction products by homochromatography [32] revealed that the major 32P-labeled digestion products generated by HSV DNase, HSV DNA polymerase and E. coli exonuclease I11 were deoxynucleoside 5‘monophosphates (Fig. 2 B). Only after longer autoradiographic exposure were oligomeric products identified among the reaction products of the HSV DNase digest, indicating that the endonucleolytic activity [24- 261 of this enzyme was very limited under the conditions for the HSV DNA polymerase reaction. Specificity of the exonucleases associated with the viral D N A polymerase and DNase
The observation that E. coli exonuclease 111 and the nucleases associated with bacterial and eukaryotic DNA polymerases are capable of degrading both strands of DNA . RNA hybrids [43, 441 led us to examine whether the exonucleases
268
:::: B
X
E
::
v
‘
10
5
40 E
O 20
1
C
2
a cu “2
2
3
4
a
b
1 2 2 a 3
1 2 3
5
6
C
1
/ 5
10
20
40
Time (min)
1 2 3
Fig. 3. Churucterizution o f t h e RNuse uctivity ussociuted with HSV DNA polymerase and HSV DNuse. (A) Kinetics of hydrolysis 3‘- and 5‘labeled RNA . DNA hybrids. Reaction mixtures (25 p1) containing 50 pg/ml 3’-[32P]UMP-labeledpoly(U) . poly(dA) (3.9 x 10’ cpm pg- 1) or 5’-[32P]UMP-labeledpoly(U) . poly(dA) (1.3 x lo5 cpm p g - l ; II), and 1 pl HSV DNA polymerase (phosphocellulose fraction; 0 )or 1 p1 HSV DNase (phosphocelluloase fraction; 0)were incubated and processed to determine the released [32P]UMPas described in the legend to Fig. 2A. (B) Degradation of p ~ l y ( [ ~ ~ P.]poly(dA) U) under RNase-H reaction conditions. Reaction mixtures were as described in Materials and Methods and aliquots (4 pl) were analyzed by homochromatography. Lane 1, template control, denatured p01y([~~P]U) . poly(dA) digested with 0.5 pg RNase A. Lane 2, 1 unit E. coli RNase H. Lane 3, 10 units Moloney murine leukemia virus reverse transcriptase. Lane 4, 0.5 pg RNase A. Lane 5 , 2 pI HSV DNA polymerase (phosphocellulose fraction). Lane 6, 2 pl HSV DNase (phosphocellulose fraction). (C) Degradation of RNA . DNA hybrids under HSV DNA polymerase reaction conditions. HSV DNA polymerase reaction mixtures (25 pl) contained alternately 50 pg/ml poly(dA) . p ~ l y ( [ ~ ~ P ](lane U ) I), p01y([~~P]A) . poly(dT) without (lane 2) or with 1 mM each of dATP and dTTP (lane 2a) and denatured p~ly([~’P]A) . poly(dT) (lane 3), and were incubated at 37°C for 30 min with either 1 p1 HSV DNA polymerase (a), 1 p1 HSV DNase (b), or 10 units E. coli exonuclease 111 (c)
Table 3. Specificity of the exonucleolytic activities of HSV DNA polymeruse, HSV DNase and E. coli exonuclease III Reaction conditions were as described in the legend to Fig. 3 C. Release of [32P]NMPfrom thc various RNA . DNA and DNA . DNA hybrids by the enzymes was determined by TLC as described, and is presented as velocities as calculated from the corresponding kinetics. The enzyme concentrations were: 1 pl HSV DNA polymerase (phosphocellulose fraction), 1 1-11 HSV DNase (phosphocellulose fraction) and 10 units E. coli exonuclease I11 (Boehringer Mannheim) Substrate
Release of [32P]NMPby exonuclease 111
polymerase
DNase
pmol . min-
. [enzyme]-
Poly(dA) . p ~ l y ( [ ~ ~ P ] d T0.47 ) Poly(dA) . p ~ l y ( [ ~ ~ P ] U0.31 ) Poly(A) . p~ly([~’P]dT) 6.0 Poly(dT) . p ~ l y ( [ ~ ~ P ] A )0.4 Poly(dT) . p ~ l y ( [ ~ ~ P ] A ) , denatured 0.4
121.8 0.52 11.7 9.7
7.15 0.16 0.2 0.72
7.3
0.6
of the viral DNA polymerase and DNase exhibited similar properties. Various synthetic 32P-labeled RNA . DNA and DNA . DNA hybrids were prepared as described in Materials and Methods, and the degradation of these substrates by the viral enzymes and E. coli exonuclease I11 was analyzed by TLC and homochromatography. As illustrated in Table 3, to
varying degrees each of the enzymes examined was capable of degrading the RNA strands of the synthetic DNA . RNA hybrids. The substrate of choice for the HSV DNase and the exonuclease I11 was the double-stranded DNA hybrid, poly(dA) . poly(dT), which was degraded at least tenfold faster than the RNA . DNA hybrids. In contrast to the latter enzymes, HSV DNA polymerase preferentially degraded the DNA strand of the RNA . DNA hybrids examined, i.e. poly(dT) was hydrolyzed 15 times faster than poly(A) using poly(A) . poly(dT) as substrate. The comparable degradation rates obtained with poly(dT) . p ~ l y ( [ ~ ~ P ]after A ) heat denaturation (Table 3) indicated that all enzymes were also capable of hydrolyzing single-stranded RNA. To determine whether the RNase activity correlated with the nuclease activities associated with viral DNA polymerase and DNase, the degradation of poly(dA) . poly(U), preferentially labeled at the 5’ or 3’ end, by these enzymes was compared. As shown in Fig. 3 A, the 3’-[32P]UMP-labeled poly(dA) . poly(U) was preferably hydrolyzed by the HSV DNA polymerase as was the 5’-labeled DNA . RNA hybrid by the HSV DNase. The major products of the RNA hydrolysis by the viral enzymes were mononucleotides even under RNase-H reaction conditions (Fig. 3 B and C). In addition oligomeric products i. e. mainly di-, tri- and tetra-meric oligoribonucleotides were formed as degradation products of poly(A) . poly(dT) by HSV DNase which revealed that both functions, exonuclease and endonuclease, of this enzyme [24 261 participated in the degradation event. In correlation with the 3’ - 5’-exonuclease function being responsible for the ob-
:z ’i!L
269
30
5
25
5
I0
20
10
20
LO
LO l 25 K 5 10 20 LO
0
T i m e (min)
L 2
4
J 6
8
Time
i 1
p.i.
l 0
1
h 2
ti,
i h )
Fig. 4. Neutralization of polymerase-associated 3’ -5’ exonuclease and 5’ - 3’ exonuclease by anti- (HSV-2DNaseJ serum. Exonuclease assays were performed with 3’-[32P]dCMP-labeled activated DNA (1.3 x lo5 cpm pg-’) under HSV DNA polymerase reaction conditions (A, B) and with 5’-[32P]poly(dT).poly(dA) (3.3 x lo5 cpm(pmo1 5’(dT) terminus) under HSV DNase reaction conditions (C, D) in the presence of either neutral rabbit serum ( 0 )or anti-(HSV-2 DNase) serum (0).Aliquots of the reaction mixtures containing 5 pl of the glycerol-gradient-purified KOS polymerase (A, C) or HSV DNase (B, D) were analyzed for released [32P]dNMPby TLC as described and the data obtained are presented as percentages of 32P-labeled added DNA
Fig. 5. Induction of HSV DNase in HSV-I KOS infected BHK-21 rells as monitored with 3‘- and 5’-32P-labeled D N A templates. Extracts of mock-( 0 )and HSV-1 KOS( 0)-infected BHK-21 cells were prepared at the indicated times after infection (p.i.) as described in text. Aliquots (1 pl) were assayed with [32P]dCMP-labeled activated DNA (50 pg/ml; 6.5 x lo4 cpm/pg) under HSV DNase standard reaction conditions (I), or with 5’-[3ZP]poly(dT). poly(dA) (50 pg/ml; 1.6 x lo5 cpm/pmol 5’4dT) terminus) under HSV DNA polymerase reaction conditions (11) for 5 min at 37‘C. Released [3ZP]dNMPwas analyzed by TLC as described in the legend to Fig. 4.DNase activity of the mock-infected extracts in experiments (I) and (11) was 1.4% and 0.02%, respectively
served RNase activity, degradation of poly(A) . poly(dT) by the HSV DNA polymerase was completely prevented in the presence of dNTP, i. e. during the ongoing polymerization reaction (Fig. 3 C).
and HSV-1-KOS-infected cells were prepared at different times after infection as described in Materials and Methods and assayed with both, 3’-[32P]dCMP-labeled activated salmon-sperm DNA under optimal HSV DNase conditions and with 5’-32P-labeled poly(dT) . poly(dA) under HSV DNA polymerase conditions. As can be seen in Fig. 5, the amount of released 5’-[32P]dNMPgenerated by the HSV DNase activity of 4-h extracts, for example, was eightfold greater with the 5’-32P-labeled substrate. In addition the background activity was found to be 70-fold lower with the latter template under conditions for the HSV DNA polymerase reaction, and demonstrated that the 5’-32P-labeledtemplate allowed more sensitive detection of HSV DNase. Taking into account that the preferred substrate is single-stranded DNA (Table l), that the specific activity of the 3’-32P-labeledactivated DNA was about twofold higher, and further that the 5’-[32P]poly(dT) consisted of more than 99% unlabeled poly(dT) in order to achieve the substrate concentration of 50 pg/ml, it is obvious that even higher sensitivity of detection of HSV DNase activity cold be realized with a 5’-labeled DNA substrate.
Neutralization of the 5‘ -3‘ exonuclease by anti-(HSV-2 DNase) serum
To prove more directly that HSV DNase contains a cryptic 5’- 3’ exonuclease, we examined whether this enzymatic activity could be neutralized by a monospecific rabbit antiserum directed against the HSV-2 DNase, which was shown to be strongly cross-reactive with the HSV-1-induced enzyme (L. M. Banks and K. Powell, personal communication). For comparison and to confirm the specificity of the antiserum, exonuclease assays were performed with the glycerol-gradientpurified viral enzymes in the presence of rabbit anti-(HSV-2 DNase) serum or control serum, and under the optimal reaction conditions for the respective enzyme. As can be seen from the results of this neutralization experiments presented in Fig. 4, HSV DNase activity was inhibited by 85% and 90% when assayed with a 3’- (Fig. 4B) and 5’-labeled substrate (Fig. 4 D), respectively. The only activity not significantly effected by the anti-(HSV-2 DNase) serum was the polymerase associated 3‘- 5’ exonuclease (Fig. 4A). Improved assay f o r H S V DNase
From the information gained so far it became clear that 5’-labeled DNA templates are the ideal substrates for detecting HSV DNase in cellular extracts. To test this, extracts of mock-
The 3‘ -5‘-exonucleolytic activity is an intrinsic function of the H S V D N A polymerase The following experimental approaches were undertaken to prove that the 3’ - 5’ exonuclease is an intrinsic property of the HSV DNA polymerase polypeptide. Thermolability of both enzymatic activities of mutant tsC7 and wild-type H S V D N A polymerase and oJ’HSV DNase. The HSV-1 KOS polymerase mutant tsC7, whose mutation was mapped to a 1.1-kbp fragment encoding the first 298 amino
270 acid residues [46], induces a DNA polymerase which is thermolabile both in vitro [35] and in vivo [l].We utilized this characteristic to ask whether the polymerase-associated 3' 5' exonuclease is also thermolabile, in order to obtain further evidence for the physical association between exonuclease and polymerase. For this purpose, the tsC7 DNA polymerase was purified from infected BHK-21 cells up to the glycerol-gradient step exactly as described for the corresponding wild-type DNA polymerase of strain KOS in Materials and Methods. Aliquots of the glycerol-gradient-purified enzymes of HSV DNase, tsC7 and KOS DNA polymerase were heat-inacti-
'"K
- ou ' A
25
'+ 2
L
1.5
10
20
lnoctivotion Time
1 2
I
L
7.5
10
min 1
Fig. 6. Thermolubility qf the exonuclease uctivities associutedwith HSV polymerase and DNase. Nine parts of enzyme in storage buffer were mixed with one part 10 mg/ml bovine serum albumin and incubated at 47' C. (A) At the stated inactivation times aliquots (10 pl) of the preincubation mixtures containing the glycerol-gradient-purified KOS ( 0 , A ) or tsC7 (0, A ) DNA polymerase were assayed both for 3'-exonuclease ( A , A )and polymerase (0,0 )activity under HSV DNA polymerase standard reaction conditions. 100% polymerase activity corresponded to an incorporation of 34.7 pmol and 19.7 pmol dNMP into activated DNA by the KOS and the tsC7 DNA polymerase. respectively. 100% 3'-exonuclease activity corresponded to the release of 21650 and 8530 acid-soluble cpm from 20 Fg/ml [32P]dAMP-labeledactivated DNA (6 x lo4 cpm pg-') by the KOS and tsC7 DNA polymerases, respectively. (B) Aliquots (10 pl) of the identically pretreated HSV DNase glycerol-gradient enzyme were assayed with 60 pg/ml [32P]dAMP-labeledactivated DNA under HSV DNase reaction conditions as described. 100% activity corresponded to 58 700 acid-soluble cpm
vated for different time intervals and then assayed for exonucleolytic and polymerizing activities under the respective optimal reaction conditions. As can be seen from Fig. 6A, the tsC7 DNA polymerase is considerably less stable than the wild-type enzyme. Furthermore, according to the inactivation profiles the polymerase and exonuclease activities of the given DNA polymerase preparations were identically inactivated. The HSV DNase activity (Fig. 6B) was more thermolabile than the exonucleolytic activity associated with the KOS polymerase. Comparison of the drug sensitivity of both enzymatic activities of mutant PAAr-5 and wild-type HSV DNA polymerase. We attempted to gain further information about the close relationship of the polymerizing and exonucleolytic functions of the HSV DNA polymerase by biochemical characterization of the drug-resistant KOS mutant PAA'-5. The PAA'-5 polymerase has recently been shown to carry a mutation at amino acid residue 842 [9] that renders this enzyme resistant to a variety of different antiviral compounds such as phosphonoacetic acid, the triphosphates of acyclovir and dideoxyguanosine and others [36, 371. To study the effect of pyrophosphate analogue upon the enzymatic activities of the PAA'-5 polymerase, the mutant enzyme was purified up to the glycerolgradient step, and its polymerizing and exonucleolytic activities were compared with those of the wild-type enzyme in the presence of phosphonoacetic acid. Fig. 7A shows that, as expected, the polymerizing activity of the PAR-5 enzyme was quite resistant to phosphonoacetic acid, and that in contrast the wild-type polymerase activity was inhibited by 80% with 20 pg/ml phosphonoacetic acid. When the effect of phosphonoacetic acid on the associated exonuclease was examined by measuring the release of 5'-[32P]dGMP from [32P]dGMPlabeled oligo(dG) . poly(dC) in the presence of 20 pg/ml phosphonoacetic acid, we found that the exonucleases exhibited drug sensitivities corresponding to the respective polymerizing activities, as shown in Fig. 7 B. In another set of experiments the effect of the chain terminator ddGTP upon both functions of wild-type and phosphonoacetic-acid-resistant polymerase was compared with that at ongoing polymerization using oligo(dG) . poly(dC) as a template. Incorporation (i. e. polymerization reaction) and excision (i. e. exonucleolytic reaction) of [32P]dGMP by the enzyme was monitored in a time course in the presence of different concentrations of ddGTP by TLC. The results of this analysis (Fig. 8) demonstrated again that exonuclease as well
B
A
-X 100 -
500
I/
z
5>
75
+ u 0
50 0 ~
10
P A A ( pg/rnl
1
20 30
40
u
50
60
10
20 30 40
50
60
T I M E ( r n l n )
Fig. 7. &ffects uf phosphonoacetic ucid on polymerizing and exonucleolytic uctivities uf wild-type KOS and PAA'-5 polymeruse. (A) Inhibition of the polymerizing activity of the KOS polymerase ( 0 )and of the PAA'-5 polymerase (A)by phosphonoacetic acid (PAA). 100% activity corresponded to the incorporation of 27 pmol and 7 pmol [32P]dAMPinto activated DNA by 2 pl KOS and PAA'-5 polymerases, respectively, under standard reaction conditions. (B) Inhibition of the 3'-exonuclease activity of KOS (I) and PAAr-5 (11) polymerases by phosphonoacetic acid. Exonuclease activity was monitored as [32P]dGMPreleased from 50 pg/ml 3'-[32P]dGMP-labeled (dG)1Z-18. poly(dC) (5.3 x lo5 cpm pg-'; 1100 cpm/pmol dGMP) by the glycerol-gradient purified KOS (4 pl) and PAAr-5 polymerase (10 pl) in the absence ( 0 ,A ) or in the presence ( 0 ,A) of 20 pg/ml phosphonoacetic acid under HSV D N A polymerase standard reaction conditions as analyzed by TLC
271 A
B
I
C
Ex30B
100
s
0
Ex306
0
EXLl
1
x I
- 50 +0
