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Inhibition of activities of DNA polymerase α, β, γ, and reverse transcriptase of L1210 cells by phosphonoacetic acid
490
Biochimica et Biophysica Acta, 520 ( 1 9 7 8 ) 4 9 0 - - 4 9 7 © Elsevier/North-Holland Biomedical Press
BBA 99270
INHIBITION OF ACTIVITIES OF DNA POLYMERASE ~, ~, ~', AND REVERSE TRANSCRIPTASE OF L1210 CELLS BY PHOSPHONOACETIC ACID
H.S. A L L A U D E E N * a n d J.R. B E R T I N O
Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.) (Received January 13th, 1978)
Summary Phosphonoacetic acid has been shown to suppress replication of DNA t u m o r viruses by inhibiting the activity of virus-induced DNA polymerase and consequently viral DNA synthesis. We now have evidence to show that phosphonoacetic acid inhibits also the cellular DNA polymerases ~, ~, and ~/of L1210 cells as well as reverse transcriptases of two type C viruses. Particularly, the DNA polymerase ~ is just as senstive as the herpes virus-induced DNA polymerase. The DNA polymerases ~ and 7 required seven times more phosphonoacetic acid for a 50% inhibition of their activities. Phosphonoacetic acid inhibited the activities of the reverse transcriptase and terminal deoxyribonucleotidyltransferase only at higher concentrations. Kinetic analysis with the DNA polymerase showed t h a t the c o m p o u n d is a non-competitive inhibitor with respect to the substrates and uncompetitive inhibitor with the activated DNA template. Studies on time course of phosphonoacetic acid inhibition revealed that the c o m p o u n d is inhibitory even after the initiation of DNA synthesis. Phosphonoacetic acid also inhibited cell growth as well as the type C virus production; at concentrations above 50 pg/ml, the inhibitory effect was more profound on the type C virus production than on cell growth.
Introduction
Sodium phosphonoacetic acid has been shown to suppress herpes virus replication both in vitro [1,2] and in vivo [3]. The antiviral effect of this compound is presumably due to its ability to suppress viral DNA replication by inhibiting the activity of virus-induced DNA polymerase [4]. The mode of * T o w h o m c o r r e s p o n d e n c e s h o u l d be addressed.
491 inhibition has been studied by Mao and Robishaw [5] and Leinbach et al. [6]. Reportedly, the c o m p o u n d brings a b o u t the inhibition b y interacting with the enzyme. The inhibition was non-competitive with dNTP substrates and uncompetitive with DNA template. Leinbach et al. [6] also suggested that the phosphonoacetic acid inhibited the reaction b y interacting with the enzyme at the pyrophosphate binding site. Mao et al. [4] and Mao and Robishaw [5] claimed that the c o m p o u n d inhibited specifically the virus-induced DNA polymerase activity. We performed experiments to evaluate the specificity of phosphonoacetic acid and found that the c o m p o u n d also inhibited the cellular DNA polymerase activities. We report here results showing that the inhibitory effect of phosphonoacetic acid is more non-specific than originally thought and that the mechanism of phosphonoacetic acid inhibition of cellular DNA polymerases is similar to that of herpes virus-induced enzymes. During the course of our study, Bolden et al. [7] reported that the DNA polymerases of HeLa cells, particularly DNA polymerase a, was also inhibited by phosphonoacetic acid, like the herpes and vaccinia virus-induced enzymes. Materials and Methods Disodium phosphonoacetate was a gift from A b b o t t Laboratories. Tritiated deoxynucleoside triphosphates and Triton X-100 were obtained from New England Nuclear Corp. Nucleoside triphosphates and synthetic primer templates were purchased from P.L. Biochemicals (Milwaukee, Wisc.). The oligonucleotide primers contained 12--18 nucleotides. Salmon sperm DNA was converted to the activated form b y treatment with DNAase I, according to the procedure of Schlabach et al. [8]. All other chemicals commercially available were of the highest purity. Cells and viruses. We used a murine lymphoid leukemia cell line L1210 (V). This cell line produces t y p e C virus particles [9]. The cells were cultured in Fischer's medium with 10% horse serum at a concentration of 104 cells/ml. The cell growth was monitored by counting the cells on each day. The t y p e C virus production was monitored by measuring the reverse transcriptase activity of the high speed (100 000 :(g) pellet of the culture medium [10]. Simian sarcoma virus was a gift from Dr. R.C. Gallo, N.I.H., Bethesda, Md. Herpes simplex virus (HSV)-induced DNA polymerase was generously provided b y Dr. A. Weissbach, Roche Institute of Molecular Biology, Nutley, N.J. Isolation of D N A polymerases and reverse transcriptase. Procedures for obtaining the DNA polymerases of L~1210 cells have been described in detail (Scanlon, K.G., Allaudeen, H.S. and Bertino, J.R., unpublished). Briefly, the cells were mixed with a solution containing 0.8 M KC1 and 0.5% Triton X-100 and disrupted using a Dounce homogenizer. Nucleic acids were removed b y passing the total cell extract through a DEAE-cellulose column which was preequilibrated in buffer A containing 50 mM Tris • HC1 (pH 7.0), 0.5 mM EDTA, 1 mM dithiothreitol, and 20% glycerol. The DNA polymerases were separated b y chromatography on a phosphocellulose column using a linear KCI gradient. The DNA polymerase ~, eluted at 0.39 M KC1, was free of other cellular DNA polymerases; the DNA polymerase a, eluted at 0.2 M KC1, contained some reverse transcriptase activity of L1210 ceils [9]. The DNA polymerase a was
492
further separated from the reverse transcriptase by chromatography on a DEAE-cellulose column with a linear KC1 gradient. Terminal deoxynucleotidyltransferase was isolated from the peripheral leukocytes of an acute lymphocytic leukemia patient [ 11 ] as previously described [ 12 ]. Reverse transcriptase from SSV and t y p e C virus produced by L1210 cells was purified according to the previously described procedure [9]. The preparations obtained after phosphocellulose chromatography were used in the assays. Enzyme assays. DNA polymerase a activity was assayed in a 50 pl standard reaction mixture which contained 50 mM Tris. HC1 (pH 7.5), 2 mM dithiothreitol, 8 mM MgC12, 100 pM each of dATP, dCTP, and dGTP, and 80 pM [aH]dTTP (450 cpm/pmol), 10 pg of activated salmon sperm DNA, and enzyme. Incubation was at 37°C for 1 h. Acid-insoluble radioactivity was collected on a Gelman nitrocellulose filter, washed several times with 5% trichloroacetic acid containing 2 mM sodium pyrophosphate and measured in a liquid scintillation counter. DNA polymerase ~ activity was assayed under similar conditions except pH 8.5 buffer and 40 mM KC1 were used. The reaction mixture for the HSV-DNA polymerase assay contained 50 mM Tris • HC1 (pH 8.0), 4 mM MgC12, and 150 mM potassium sulfate. Other ingredients are the same. The reaction mixture for the assay of reverse transcriptase activity contained the following ingredients in a total volume of 50 pl: 50 mM Tris • HC1 buffer (pH 8.0), 1 mM MnC12, 80 mM KC1, 2 mM dithiothreitol, 30 pM dATP, 20 uM [3H]dGTP (1800 cpm/pmol), 0.5 pg of (dG)~~s" (cm)n and enzyme. Terminal deoxynucleotidyltransferase activity was assayed in a 50 pl reaction mixture which contained 50 mM Tris. HC1 (pH 8.0), 2 mM dithiothreitol, 0.6 mM MnCl:, 2 pg of (dA)~lS, 200 pM of tritiated dGTP (300 cpm/ pmol) and enzyme. Results
Effect of phosphonoacetic acid on the activities of DNA polymerases. The inhibition of DNA polymerases by phosphonoacetic acid is shown in Fig. 1. Of 100
10-5
10-4
10-5
I 10-2
PAA (M)
Fig. 1. P h o s p h o n o a c e t i c acid i n h i b i t i o n o f v a r i o u s D N A p o l y m e r a s e s . P e r c e n t a g e o f t h e r e m a i n i n g a c t i v i t y w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f p h o s p h o n o a c e t i c acid is s h o w n . D N A p o l y m e r a s e s ~, ~. a n d 3", a n d H S V D N A p o l y m e r a s e c o n t a i n e d 4 2 , 3 3 , 7, a n d 4 u n i t s o f t h e e n z y m e in e a c h e x p e r i m e n t , r e s p e c t i v e l y . Activ a t e d D N A was u s e d as t h e t e m p l a t e ; o t h e r c o n d i t i o n s o f t h e assay are d e s c r i b e d in t h e t e x t .
493 TABLE I INHIBITION OF DNA POLYMERASES
BY PHOSPHONOACETIC
ACID
The enzyme units represent pmol of the corresponding dNMP incorporated at 37°C for 60 min under optim u m a s s a y c o n d i t i o n s . T h e D N A p o l y m e r a s e s ~, fl, a n d 3, f r o m L 1 2 1 0 cells w e r e u s e d ; 1 0 0 % a c t i v i t y o f these enzymes represent 53, 42, and 11 units, respectively. 100% activity of HSV-DNA polymerase (HSVDP), S S V - r e v e r s e t r a n s c r i p t a s e , ( S S V - R T ) . L 1 2 1 0 - r e v e r s e t r a n s c r i p t a s e ( L 1 2 1 0 - R T ) a n d t e r m i n a l d e o x y nucleotidyltransferase (TdT) represent 9, 27, 31, and 7 units of activity, respectively. Other assay conditions are described in the text. Phospbono-
I n h i b i t i o n o f D N A p o l y m e r a s e a c t i v i t y (%)
acetic acid
( X 1 0 -4 M)
~
fl
7
HSV-DP
SSV-RT
L1210 RT
TdT
0.5 1 2 10
61.1 74.2 81.7 94.5
31.0 45.0 46.0 87.0
24.5 46.2 47.0 75
69.5 81.0 82.3 98.0
47.3 64.0 73.0 82.2
32.0 62.0 61.4 79.0
29.0 57.1 59.1 62.0
the cellular enzymes, DNA polymerase ~ was most sensitive to phosphonoacetic acid inhibition, and DNA polymerase fl and 7 were least sensitive. HSVinduced DNA polymerase, used as a control, was slightly more sensitive than DNA polymerase ~. Phosphonoacetic acid was inhibitory to n o t only DNAdependent DNA polymerase activity but RNA-dependent DNA polymerase as well as terminal deoxynucleotidyltransferase activities were also affected (Table I). However, phosphonoacetic acid inhibited the activities of these enzymes only weakly. For example, the phosphonoacetic acid concentrations required for 50% inhibition of the activities of reverse transcriptase of SSV, and terminal deoxynucleotidyltransferase of acute l y m p h o c y t i c leukemia leukocytes are 7 • 10-4 and 8 • 10-4 M, respectively; the activities of DNA polymerase ~ and HSV-induced DNA polymerase were inhibited 50% by approx. 2.5 • 10 -s M of phosphonoacetic acid. Time course o f phosphonoacetic acid inhibition. To determine whether 40
ONA POLYMERASE /~
DNA POLYMERASE ¢1
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60 TIME
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Fig. 2. T i m e c o u r s e o f p h o s p h o n o a c e t i c a c i d i n h i b i t i o n . P h o s p h o n o a c e t i c a c i d a t a f i n a l c o n c e n t r a t i o n o f 5 • 1 0 -4 M w a s a d d e d a t t h e t i m e i n t e r v a l as s h o w n a f t e r t h e i n i t i a t i o n o f t h e e n z y m e r e a c t i o n . T r i t i a t e d d G T P ( 4 5 0 c p m / p m o l ) w a s u s e d in t h i s e x p e r i m e n t ; all t h e o t h e r c o n d i t i o n s a r e d e s c r i b e d in t h e t e x t .
494 DNA POLYMERASE 007
DNA POLYMERASE ,~
PAA IOx qO-5M [3
0.12
005
-
PAA 5x10-4 M
006
a.
PAA 5 x 10"SM
E a. v
0.0
o
0.10 008 PAA IxIO'4M
~-
O0
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0.06
O0
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002
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OI
-01
0.2
03
0.4
-01
NO PAA
O,
02
013
I
I
dTTP(/.~M)
dTTP (;zM)
04
Figs. 3 and 4. Effect of phosphonoacetic a c i d o n t h e r e a c t i o n r a t e in t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a tions of dTTP with DNA polymerase a (Fig. 3) and DNA polymerase/3 (Fig. 4).
phosphonoacetic acid could inhibit DNA synthesis even after the reaction was initiated, it was added to the ongoing reaction system at different time intervals and the activity was monitored. As shown in Fig. 2, the compound caused an instantaneous inhibition whether the drug was added at the time of initiation or after the initiation of the reaction. Effect of substrate concentration on phosphonoacetic acid inhibition. To characterize further the nature of the inhibition, we examined phosphonoacetic acid inhibition with increasing substrate concentration. Tritiated dTTP was used as the rate-limiting substrate, the other three triphosphates were in excess. When the data were plotted by the method of Lineweaver and Burk [13], straigth lines could be drawn intersecting on the abscissa. These plots indicate that the inhibition was n o n c o m p e t i t i v e with dTTP (Fig. 3). The apparent Km value of DNA polymerase a for [3H]dTTP was 5.5 pM; the Ki for phosphonoacetic acid was approximately four times higher than the Kin. A similar pattern of inhibition was observed with DNA polymerase fl; however, higher concentraONA POLYMERASE n
A
0. I0
o E a. 0.08 uJ I< 0c o Q.
0.06
0
0,04
0 PAA I x l O ' 4 M
nr Z 0.02
I
1
-0.1
0.1
I
I
1
0.2
0.3
0.4
DNA (lz,g) Fig. 5. Effect of phosphonoacetic activated DNA.
acid on the reaction rate in the presence of different concentrations
of
495 T A B L E II PHOSPHONOACETIC ACID INHIBITION WITH DIFFERENT SYNTHETIC PRIMER TEMPLATES F o r l e g e n d see T a b l e I. Enzyme
DNA polymerase ~
DNA polymerase ~
Phosphonoacetic acid addition
I n h i b i t i o n o f D N A p o l y m e r a s e a c t i v i t y (%)
(Xl0 -4 M )
DNA
0 1
0 74.8
0 97.9
--
2
81.7
98.9
--
0 2 5
0 45.1 76.0
0 24.8 49.1
Activated
(dT)~15 • (dA)n
( d T ) ~ 1 5 • (A)n
--
0 26.2 31.1
tions of phosphonoacetic acid were required for inhibition (Fig. 4). The apparent Km value of DNA polymerase t3 for dTTP was 14 pM; the K i for phosphonoacetic acid was approx. 18 times more than the Km. When the experiments were repeated using, alternatively, radioactive dATP, dCTP, or dGTP as the rate-limiting substrates, similar results were obtained. Effect of activated DNA concentration on phosphonoacetic acid inhibition• As shown in Fig. 5, the double reciprocal plots of DNA synthesis vs. template concentration at various concentrations of phosphonoacetic acid yielded parallel lines indicating that the inhibition was uncompetitive with the template DNA. Similar experiments with DNA polymerase/~ yielded similar results. Phosphonoacetic acid inhibition with synthetic primer templates in the assay. The extent of inhibition b y phosphonoacetic acid when different synthetic primer templates were used in the assay was tested. These studies showed that change of templates did n o t have a profound effect on the nature of phos-
106
50/z9 control 100m
~
zoom
~, io5 d
, °,
I
2
4
5
6
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~AYS Fig• 6. E f f e c t o f p h o s p h o n o a c e f i c acid o n t h e g r o w t h o f L 1 2 1 0 cells. The ten g r o w t h was m o n i t o r e d d a i l y
using a C o u l t e r c o u n t e r . T h e c o n c e n t r a t i o n s (~tg/ml) of p h o s P h o n o a c e t i c acid a d d e d are s h o w n . T h e t y p e C v i r u s p r o d u c t i o n w a s m o n i t o r e d b y e s t i m a t i n g t h e r e v e r s e t r a n s c r i p t a s e a c t i v i t y of t h e 1 0 0 0 0 0 X g p e l l e t of t h e c u l t u r e s u p e r n a t a n t as d e s c r i b e d p r e v / o u s l y [ 1 0 ] . Solid b a r s r e p r e s e n t t h e d e c r e a s e in t h e virus p r o d u c t i o n in t h e p r e s e n c e of 50 pg of p h o s p h o n o a c e t i c acid.
496 phonoacetic acid inhibition. However, the difference in sensitivity to phosphonoacetic acid between DNA polymerase ~ and DNA polymerase fi was greater when the synthetic primer templates were used in place of activated DNA (Table II). Effect o f phosphonoacetic acid on growth of L1210 cells and type C virus production. The effect of phosphonoacetic acid on the growth of L1210 cells and the production of type C virus was examined. As shown in Fig. 6, phosphonoacetic acid at 50 pg/ml did n o t have any noticeable effect on cell growth; however, higher concentrations of phosphonoacetic acid reduced growth considerably. Phosphonoacetic acid had a more profound effect on the type C virus production than on cell growth; for example, at 50 pg/ml on day 3, the virus production was inhibited more than 50% while cell growth was affected only b y 16%. Discussion
Phosphonoacetic acid is a small molecule which inhibits reversibly the multiplication of several herpes and vaccinia viruses. The c o m p o u n d inhibits viral DNA replication b y inhibiting the virus-induced DNA polymerase. Although the inhibitory effect of phosphonoacetic acid on the herpes virus-induced enzymes is impressive, it is n o t specific to herpes virus enzymes as originally reported from studies with WI-38 DNA polymerases [5]. The DNA polymerase of HeLa cells [7], duck e m b r y o fibroblasts [6], and horse t u m o r cells [14] was also inhibited. Our results show that the DNA polymerase ~ of L1210 cells is just as sensitive as the herpes virus-induced DNA polymerase. However, the DNA polymerase fi and 7 required seven times more phosphonoacetic acid for a 50% inhibition of their activities. The inhibition pattern was similar when different primer templates were used in the enzyme assays indicating that the template is n o t the likely site of action. At higher concentrations, the c o m p o u n d can also inhibit the activities of reverse transcriptases of SSV and L1210 virus as well as terminal deoxynucleotidyltransferase of an acute lymphocytic leukemia patient. Kinetic analysis with the DNA polymerase ~ showed that the comp o u n d is a non-competitive inhibitor with the activated DNA. These results are consistent with the mode of inhibition of phosphonoacetic acid observed with herpes virus-induced DNA polymerases [5,6]. Leinbach et al. [6] have proposed that the phosphonoacetic acid binds to the polymerase at the pyrophosphate binding site and is, thus, a competitive inhibitor of pyrophosphate in the exchange reaction. However, future verification of this interesting scheme may require identification of the postulated phosphonoacetic acid b o u n d intermediate. Acknowledgements We thank Mr. S.A. Hasan for technical assistance. This research was supported b y Grant CH-47 of the American Cancer Society and CA 08010 and 08341 of the United States Public Health Service.
497
References 1 0 v e r b y , L.R., Robishaw, E.E., Schleicher, J.B., Rueter, A., Shipkowitz, N.L. and Mao, J.C.-H. (1974) Antimicrob. Agents Chemother. 6 , 3 6 0 - - 3 6 5 2 Yajima, Y., Tanaka, A. and Nonoyama, M. (1976) Virology 7 1 , 3 5 2 - - 3 5 4 3 Shipkowitz, N.L., Bower, R.R., Appell, R.N., Nordeen, C.W., Overby, L.R., Roderick, W.R., Schleicher, J.B. and Von Esch, A.M. (1973) Appl. Mierobiol. 2 6 , 2 6 4 - - 2 6 7 4 Mao, J.C.-H., Robishaw, E.E. and Overby, L.R. (1975) J. Virol. 15, 1281--1283 5 Mao, J.C.-H. and Robishaw, E.E. (1975) Biochemistry 14, 5475--5479 6 Leinbach, S.S., Reno, J.M., Lee, L.F., Isbell, A.F. and Boezi, J.A. (1976) Biochemistry 15, 426--430 7 Bolden, A., Aucker, J. and Weissbach, A. (1975) J. ViroL 16, 1584--1592 8 Schlabach, A., Fridlander, A., Bolden, A. and Weissbach, A. (1971) Biochem. Biophys. Res. Commun. 44, 879--885 9 Allaudeen, H.S, and Bertino, J.R. (1977) J. Natl. Cancer Inst. 5 9 , 2 2 7 - - 2 3 5 10 Allaudeen, H.S., Sarngadharan, M.G. and Gallo, R.C. (1976) Bioehim. Biophys. Acta 435, 45--62 11 Shaw, M.T., Dwyer, J.M., Allaudeen, H.S. and Weitzman, H.A. (1978) Blood 5 1 , 1 8 1 - - 1 8 7 12 Satin, P.S., Anderson, P.N. and Gallo, R.C. (1976) Blood 47, 11--20 13 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 56, 658--666 14 Allen, G.P., O'Callaghan, J. and Randall, C.C. (1977) Virology 76. 395--408
Biochimica et Biophysica Acta, 520 ( 1 9 7 8 ) 4 9 0 - - 4 9 7 © Elsevier/North-Holland Biomedical Press
BBA 99270
INHIBITION OF ACTIVITIES OF DNA POLYMERASE ~, ~, ~', AND REVERSE TRANSCRIPTASE OF L1210 CELLS BY PHOSPHONOACETIC ACID
H.S. A L L A U D E E N * a n d J.R. B E R T I N O
Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.) (Received January 13th, 1978)
Summary Phosphonoacetic acid has been shown to suppress replication of DNA t u m o r viruses by inhibiting the activity of virus-induced DNA polymerase and consequently viral DNA synthesis. We now have evidence to show that phosphonoacetic acid inhibits also the cellular DNA polymerases ~, ~, and ~/of L1210 cells as well as reverse transcriptases of two type C viruses. Particularly, the DNA polymerase ~ is just as senstive as the herpes virus-induced DNA polymerase. The DNA polymerases ~ and 7 required seven times more phosphonoacetic acid for a 50% inhibition of their activities. Phosphonoacetic acid inhibited the activities of the reverse transcriptase and terminal deoxyribonucleotidyltransferase only at higher concentrations. Kinetic analysis with the DNA polymerase showed t h a t the c o m p o u n d is a non-competitive inhibitor with respect to the substrates and uncompetitive inhibitor with the activated DNA template. Studies on time course of phosphonoacetic acid inhibition revealed that the c o m p o u n d is inhibitory even after the initiation of DNA synthesis. Phosphonoacetic acid also inhibited cell growth as well as the type C virus production; at concentrations above 50 pg/ml, the inhibitory effect was more profound on the type C virus production than on cell growth.
Introduction
Sodium phosphonoacetic acid has been shown to suppress herpes virus replication both in vitro [1,2] and in vivo [3]. The antiviral effect of this compound is presumably due to its ability to suppress viral DNA replication by inhibiting the activity of virus-induced DNA polymerase [4]. The mode of * T o w h o m c o r r e s p o n d e n c e s h o u l d be addressed.
491 inhibition has been studied by Mao and Robishaw [5] and Leinbach et al. [6]. Reportedly, the c o m p o u n d brings a b o u t the inhibition b y interacting with the enzyme. The inhibition was non-competitive with dNTP substrates and uncompetitive with DNA template. Leinbach et al. [6] also suggested that the phosphonoacetic acid inhibited the reaction b y interacting with the enzyme at the pyrophosphate binding site. Mao et al. [4] and Mao and Robishaw [5] claimed that the c o m p o u n d inhibited specifically the virus-induced DNA polymerase activity. We performed experiments to evaluate the specificity of phosphonoacetic acid and found that the c o m p o u n d also inhibited the cellular DNA polymerase activities. We report here results showing that the inhibitory effect of phosphonoacetic acid is more non-specific than originally thought and that the mechanism of phosphonoacetic acid inhibition of cellular DNA polymerases is similar to that of herpes virus-induced enzymes. During the course of our study, Bolden et al. [7] reported that the DNA polymerases of HeLa cells, particularly DNA polymerase a, was also inhibited by phosphonoacetic acid, like the herpes and vaccinia virus-induced enzymes. Materials and Methods Disodium phosphonoacetate was a gift from A b b o t t Laboratories. Tritiated deoxynucleoside triphosphates and Triton X-100 were obtained from New England Nuclear Corp. Nucleoside triphosphates and synthetic primer templates were purchased from P.L. Biochemicals (Milwaukee, Wisc.). The oligonucleotide primers contained 12--18 nucleotides. Salmon sperm DNA was converted to the activated form b y treatment with DNAase I, according to the procedure of Schlabach et al. [8]. All other chemicals commercially available were of the highest purity. Cells and viruses. We used a murine lymphoid leukemia cell line L1210 (V). This cell line produces t y p e C virus particles [9]. The cells were cultured in Fischer's medium with 10% horse serum at a concentration of 104 cells/ml. The cell growth was monitored by counting the cells on each day. The t y p e C virus production was monitored by measuring the reverse transcriptase activity of the high speed (100 000 :(g) pellet of the culture medium [10]. Simian sarcoma virus was a gift from Dr. R.C. Gallo, N.I.H., Bethesda, Md. Herpes simplex virus (HSV)-induced DNA polymerase was generously provided b y Dr. A. Weissbach, Roche Institute of Molecular Biology, Nutley, N.J. Isolation of D N A polymerases and reverse transcriptase. Procedures for obtaining the DNA polymerases of L~1210 cells have been described in detail (Scanlon, K.G., Allaudeen, H.S. and Bertino, J.R., unpublished). Briefly, the cells were mixed with a solution containing 0.8 M KC1 and 0.5% Triton X-100 and disrupted using a Dounce homogenizer. Nucleic acids were removed b y passing the total cell extract through a DEAE-cellulose column which was preequilibrated in buffer A containing 50 mM Tris • HC1 (pH 7.0), 0.5 mM EDTA, 1 mM dithiothreitol, and 20% glycerol. The DNA polymerases were separated b y chromatography on a phosphocellulose column using a linear KCI gradient. The DNA polymerase ~, eluted at 0.39 M KC1, was free of other cellular DNA polymerases; the DNA polymerase a, eluted at 0.2 M KC1, contained some reverse transcriptase activity of L1210 ceils [9]. The DNA polymerase a was
492
further separated from the reverse transcriptase by chromatography on a DEAE-cellulose column with a linear KC1 gradient. Terminal deoxynucleotidyltransferase was isolated from the peripheral leukocytes of an acute lymphocytic leukemia patient [ 11 ] as previously described [ 12 ]. Reverse transcriptase from SSV and t y p e C virus produced by L1210 cells was purified according to the previously described procedure [9]. The preparations obtained after phosphocellulose chromatography were used in the assays. Enzyme assays. DNA polymerase a activity was assayed in a 50 pl standard reaction mixture which contained 50 mM Tris. HC1 (pH 7.5), 2 mM dithiothreitol, 8 mM MgC12, 100 pM each of dATP, dCTP, and dGTP, and 80 pM [aH]dTTP (450 cpm/pmol), 10 pg of activated salmon sperm DNA, and enzyme. Incubation was at 37°C for 1 h. Acid-insoluble radioactivity was collected on a Gelman nitrocellulose filter, washed several times with 5% trichloroacetic acid containing 2 mM sodium pyrophosphate and measured in a liquid scintillation counter. DNA polymerase ~ activity was assayed under similar conditions except pH 8.5 buffer and 40 mM KC1 were used. The reaction mixture for the HSV-DNA polymerase assay contained 50 mM Tris • HC1 (pH 8.0), 4 mM MgC12, and 150 mM potassium sulfate. Other ingredients are the same. The reaction mixture for the assay of reverse transcriptase activity contained the following ingredients in a total volume of 50 pl: 50 mM Tris • HC1 buffer (pH 8.0), 1 mM MnC12, 80 mM KC1, 2 mM dithiothreitol, 30 pM dATP, 20 uM [3H]dGTP (1800 cpm/pmol), 0.5 pg of (dG)~~s" (cm)n and enzyme. Terminal deoxynucleotidyltransferase activity was assayed in a 50 pl reaction mixture which contained 50 mM Tris. HC1 (pH 8.0), 2 mM dithiothreitol, 0.6 mM MnCl:, 2 pg of (dA)~lS, 200 pM of tritiated dGTP (300 cpm/ pmol) and enzyme. Results
Effect of phosphonoacetic acid on the activities of DNA polymerases. The inhibition of DNA polymerases by phosphonoacetic acid is shown in Fig. 1. Of 100
10-5
10-4
10-5
I 10-2
PAA (M)
Fig. 1. P h o s p h o n o a c e t i c acid i n h i b i t i o n o f v a r i o u s D N A p o l y m e r a s e s . P e r c e n t a g e o f t h e r e m a i n i n g a c t i v i t y w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f p h o s p h o n o a c e t i c acid is s h o w n . D N A p o l y m e r a s e s ~, ~. a n d 3", a n d H S V D N A p o l y m e r a s e c o n t a i n e d 4 2 , 3 3 , 7, a n d 4 u n i t s o f t h e e n z y m e in e a c h e x p e r i m e n t , r e s p e c t i v e l y . Activ a t e d D N A was u s e d as t h e t e m p l a t e ; o t h e r c o n d i t i o n s o f t h e assay are d e s c r i b e d in t h e t e x t .
493 TABLE I INHIBITION OF DNA POLYMERASES
BY PHOSPHONOACETIC
ACID
The enzyme units represent pmol of the corresponding dNMP incorporated at 37°C for 60 min under optim u m a s s a y c o n d i t i o n s . T h e D N A p o l y m e r a s e s ~, fl, a n d 3, f r o m L 1 2 1 0 cells w e r e u s e d ; 1 0 0 % a c t i v i t y o f these enzymes represent 53, 42, and 11 units, respectively. 100% activity of HSV-DNA polymerase (HSVDP), S S V - r e v e r s e t r a n s c r i p t a s e , ( S S V - R T ) . L 1 2 1 0 - r e v e r s e t r a n s c r i p t a s e ( L 1 2 1 0 - R T ) a n d t e r m i n a l d e o x y nucleotidyltransferase (TdT) represent 9, 27, 31, and 7 units of activity, respectively. Other assay conditions are described in the text. Phospbono-
I n h i b i t i o n o f D N A p o l y m e r a s e a c t i v i t y (%)
acetic acid
( X 1 0 -4 M)
~
fl
7
HSV-DP
SSV-RT
L1210 RT
TdT
0.5 1 2 10
61.1 74.2 81.7 94.5
31.0 45.0 46.0 87.0
24.5 46.2 47.0 75
69.5 81.0 82.3 98.0
47.3 64.0 73.0 82.2
32.0 62.0 61.4 79.0
29.0 57.1 59.1 62.0
the cellular enzymes, DNA polymerase ~ was most sensitive to phosphonoacetic acid inhibition, and DNA polymerase fl and 7 were least sensitive. HSVinduced DNA polymerase, used as a control, was slightly more sensitive than DNA polymerase ~. Phosphonoacetic acid was inhibitory to n o t only DNAdependent DNA polymerase activity but RNA-dependent DNA polymerase as well as terminal deoxynucleotidyltransferase activities were also affected (Table I). However, phosphonoacetic acid inhibited the activities of these enzymes only weakly. For example, the phosphonoacetic acid concentrations required for 50% inhibition of the activities of reverse transcriptase of SSV, and terminal deoxynucleotidyltransferase of acute l y m p h o c y t i c leukemia leukocytes are 7 • 10-4 and 8 • 10-4 M, respectively; the activities of DNA polymerase ~ and HSV-induced DNA polymerase were inhibited 50% by approx. 2.5 • 10 -s M of phosphonoacetic acid. Time course o f phosphonoacetic acid inhibition. To determine whether 40
ONA POLYMERASE /~
DNA POLYMERASE ¢1
55
o.
so
// //
~ z5
[]
0:
o 20 0
z~
t5 ////"
/ 15
30
45
60 TIME
//G
I
I
I
I
15
30
45
60
minute)
Fig. 2. T i m e c o u r s e o f p h o s p h o n o a c e t i c a c i d i n h i b i t i o n . P h o s p h o n o a c e t i c a c i d a t a f i n a l c o n c e n t r a t i o n o f 5 • 1 0 -4 M w a s a d d e d a t t h e t i m e i n t e r v a l as s h o w n a f t e r t h e i n i t i a t i o n o f t h e e n z y m e r e a c t i o n . T r i t i a t e d d G T P ( 4 5 0 c p m / p m o l ) w a s u s e d in t h i s e x p e r i m e n t ; all t h e o t h e r c o n d i t i o n s a r e d e s c r i b e d in t h e t e x t .
494 DNA POLYMERASE 007
DNA POLYMERASE ,~
PAA IOx qO-5M [3
0.12
005
-
PAA 5x10-4 M
006
a.
PAA 5 x 10"SM
E a. v
0.0
o
0.10 008 PAA IxIO'4M
~-
O0
a_
0.06
O0
o 2: o_
0O4
-o
002
NO PAA
OI
-01
0.2
03
0.4
-01
NO PAA
O,
02
013
I
I
dTTP(/.~M)
dTTP (;zM)
04
Figs. 3 and 4. Effect of phosphonoacetic a c i d o n t h e r e a c t i o n r a t e in t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a tions of dTTP with DNA polymerase a (Fig. 3) and DNA polymerase/3 (Fig. 4).
phosphonoacetic acid could inhibit DNA synthesis even after the reaction was initiated, it was added to the ongoing reaction system at different time intervals and the activity was monitored. As shown in Fig. 2, the compound caused an instantaneous inhibition whether the drug was added at the time of initiation or after the initiation of the reaction. Effect of substrate concentration on phosphonoacetic acid inhibition. To characterize further the nature of the inhibition, we examined phosphonoacetic acid inhibition with increasing substrate concentration. Tritiated dTTP was used as the rate-limiting substrate, the other three triphosphates were in excess. When the data were plotted by the method of Lineweaver and Burk [13], straigth lines could be drawn intersecting on the abscissa. These plots indicate that the inhibition was n o n c o m p e t i t i v e with dTTP (Fig. 3). The apparent Km value of DNA polymerase a for [3H]dTTP was 5.5 pM; the Ki for phosphonoacetic acid was approximately four times higher than the Kin. A similar pattern of inhibition was observed with DNA polymerase fl; however, higher concentraONA POLYMERASE n
A
0. I0
o E a. 0.08 uJ I< 0c o Q.
0.06
0
0,04
0 PAA I x l O ' 4 M
nr Z 0.02
I
1
-0.1
0.1
I
I
1
0.2
0.3
0.4
DNA (lz,g) Fig. 5. Effect of phosphonoacetic activated DNA.
acid on the reaction rate in the presence of different concentrations
of
495 T A B L E II PHOSPHONOACETIC ACID INHIBITION WITH DIFFERENT SYNTHETIC PRIMER TEMPLATES F o r l e g e n d see T a b l e I. Enzyme
DNA polymerase ~
DNA polymerase ~
Phosphonoacetic acid addition
I n h i b i t i o n o f D N A p o l y m e r a s e a c t i v i t y (%)
(Xl0 -4 M )
DNA
0 1
0 74.8
0 97.9
--
2
81.7
98.9
--
0 2 5
0 45.1 76.0
0 24.8 49.1
Activated
(dT)~15 • (dA)n
( d T ) ~ 1 5 • (A)n
--
0 26.2 31.1
tions of phosphonoacetic acid were required for inhibition (Fig. 4). The apparent Km value of DNA polymerase t3 for dTTP was 14 pM; the K i for phosphonoacetic acid was approx. 18 times more than the Km. When the experiments were repeated using, alternatively, radioactive dATP, dCTP, or dGTP as the rate-limiting substrates, similar results were obtained. Effect of activated DNA concentration on phosphonoacetic acid inhibition• As shown in Fig. 5, the double reciprocal plots of DNA synthesis vs. template concentration at various concentrations of phosphonoacetic acid yielded parallel lines indicating that the inhibition was uncompetitive with the template DNA. Similar experiments with DNA polymerase/~ yielded similar results. Phosphonoacetic acid inhibition with synthetic primer templates in the assay. The extent of inhibition b y phosphonoacetic acid when different synthetic primer templates were used in the assay was tested. These studies showed that change of templates did n o t have a profound effect on the nature of phos-
106
50/z9 control 100m
~
zoom
~, io5 d
, °,
I
2
4
5
6
>
~AYS Fig• 6. E f f e c t o f p h o s p h o n o a c e f i c acid o n t h e g r o w t h o f L 1 2 1 0 cells. The ten g r o w t h was m o n i t o r e d d a i l y
using a C o u l t e r c o u n t e r . T h e c o n c e n t r a t i o n s (~tg/ml) of p h o s P h o n o a c e t i c acid a d d e d are s h o w n . T h e t y p e C v i r u s p r o d u c t i o n w a s m o n i t o r e d b y e s t i m a t i n g t h e r e v e r s e t r a n s c r i p t a s e a c t i v i t y of t h e 1 0 0 0 0 0 X g p e l l e t of t h e c u l t u r e s u p e r n a t a n t as d e s c r i b e d p r e v / o u s l y [ 1 0 ] . Solid b a r s r e p r e s e n t t h e d e c r e a s e in t h e virus p r o d u c t i o n in t h e p r e s e n c e of 50 pg of p h o s p h o n o a c e t i c acid.
496 phonoacetic acid inhibition. However, the difference in sensitivity to phosphonoacetic acid between DNA polymerase ~ and DNA polymerase fi was greater when the synthetic primer templates were used in place of activated DNA (Table II). Effect o f phosphonoacetic acid on growth of L1210 cells and type C virus production. The effect of phosphonoacetic acid on the growth of L1210 cells and the production of type C virus was examined. As shown in Fig. 6, phosphonoacetic acid at 50 pg/ml did n o t have any noticeable effect on cell growth; however, higher concentrations of phosphonoacetic acid reduced growth considerably. Phosphonoacetic acid had a more profound effect on the type C virus production than on cell growth; for example, at 50 pg/ml on day 3, the virus production was inhibited more than 50% while cell growth was affected only b y 16%. Discussion
Phosphonoacetic acid is a small molecule which inhibits reversibly the multiplication of several herpes and vaccinia viruses. The c o m p o u n d inhibits viral DNA replication b y inhibiting the virus-induced DNA polymerase. Although the inhibitory effect of phosphonoacetic acid on the herpes virus-induced enzymes is impressive, it is n o t specific to herpes virus enzymes as originally reported from studies with WI-38 DNA polymerases [5]. The DNA polymerase of HeLa cells [7], duck e m b r y o fibroblasts [6], and horse t u m o r cells [14] was also inhibited. Our results show that the DNA polymerase ~ of L1210 cells is just as sensitive as the herpes virus-induced DNA polymerase. However, the DNA polymerase fi and 7 required seven times more phosphonoacetic acid for a 50% inhibition of their activities. The inhibition pattern was similar when different primer templates were used in the enzyme assays indicating that the template is n o t the likely site of action. At higher concentrations, the c o m p o u n d can also inhibit the activities of reverse transcriptases of SSV and L1210 virus as well as terminal deoxynucleotidyltransferase of an acute lymphocytic leukemia patient. Kinetic analysis with the DNA polymerase ~ showed that the comp o u n d is a non-competitive inhibitor with the activated DNA. These results are consistent with the mode of inhibition of phosphonoacetic acid observed with herpes virus-induced DNA polymerases [5,6]. Leinbach et al. [6] have proposed that the phosphonoacetic acid binds to the polymerase at the pyrophosphate binding site and is, thus, a competitive inhibitor of pyrophosphate in the exchange reaction. However, future verification of this interesting scheme may require identification of the postulated phosphonoacetic acid b o u n d intermediate. Acknowledgements We thank Mr. S.A. Hasan for technical assistance. This research was supported b y Grant CH-47 of the American Cancer Society and CA 08010 and 08341 of the United States Public Health Service.
497
References 1 0 v e r b y , L.R., Robishaw, E.E., Schleicher, J.B., Rueter, A., Shipkowitz, N.L. and Mao, J.C.-H. (1974) Antimicrob. Agents Chemother. 6 , 3 6 0 - - 3 6 5 2 Yajima, Y., Tanaka, A. and Nonoyama, M. (1976) Virology 7 1 , 3 5 2 - - 3 5 4 3 Shipkowitz, N.L., Bower, R.R., Appell, R.N., Nordeen, C.W., Overby, L.R., Roderick, W.R., Schleicher, J.B. and Von Esch, A.M. (1973) Appl. Mierobiol. 2 6 , 2 6 4 - - 2 6 7 4 Mao, J.C.-H., Robishaw, E.E. and Overby, L.R. (1975) J. Virol. 15, 1281--1283 5 Mao, J.C.-H. and Robishaw, E.E. (1975) Biochemistry 14, 5475--5479 6 Leinbach, S.S., Reno, J.M., Lee, L.F., Isbell, A.F. and Boezi, J.A. (1976) Biochemistry 15, 426--430 7 Bolden, A., Aucker, J. and Weissbach, A. (1975) J. ViroL 16, 1584--1592 8 Schlabach, A., Fridlander, A., Bolden, A. and Weissbach, A. (1971) Biochem. Biophys. Res. Commun. 44, 879--885 9 Allaudeen, H.S, and Bertino, J.R. (1977) J. Natl. Cancer Inst. 5 9 , 2 2 7 - - 2 3 5 10 Allaudeen, H.S., Sarngadharan, M.G. and Gallo, R.C. (1976) Bioehim. Biophys. Acta 435, 45--62 11 Shaw, M.T., Dwyer, J.M., Allaudeen, H.S. and Weitzman, H.A. (1978) Blood 5 1 , 1 8 1 - - 1 8 7 12 Satin, P.S., Anderson, P.N. and Gallo, R.C. (1976) Blood 47, 11--20 13 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 56, 658--666 14 Allen, G.P., O'Callaghan, J. and Randall, C.C. (1977) Virology 76. 395--408