Differential effects of aphidicolin on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells

413

Biochimica et Biophysica Acta, 610 (1980) 413--420

© Elsevier/North-HollandBiomedicalPress

BBA 9 9 7 9 6

DIFFERENTIAL EFFECTS OF APHIDICOLIN ON REPLICATIVE DNA SYNTHESIS AND UNSCHEDULED DNA SYNTHESIS IN PERMEABLE MOUSE SARCOMA CELLS

SHUJI SEKI a, T A K U Z O

O D A a and M O C H I H I K O O H A S H I b

a Department of Biochemistry, Cancer Institute,Okayama UniversityMedical School 2-5-1,Shikata-eho, Okayama 700 and b Department of Biochemistry, Tokyo Metropolitan Instituteof Gerontology, Sahae-cho, Itabashi-ku,Tokyo 173 (Japan) (Received May 8th, 1980)

Key words: Aphidicolin; DNA synthesis; DNA polymerase; (Mouse sarcoma cell)

Summary Aphidicolin dearly discriminated replicative DNA synthesis from unscheduled DNA synthesis. Aphidicolin inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells. The mode of inhibition of aphidicolin was a mixed type with respect to deoxycytidine triphosphate but was non,competitive with respect to the other three deoxynucleoside triphosphates. Aphidicolin did not affect the activity of unscheduled DNA synthesis in either bleomycintreated permeable sarcoma cells or isolated rat liver nuclei. Considering the difference in sensitivity of DNA polymerase a and fi to aphidicolin, and other related information reported previously, the results are compatible with the idea that DNA polymerase a is involved in replicative DNA synthesis and DNA polymerase fi in unscheduled DNA synthesis in the present systems.

Introduction DNA polymerases in mammalian cells are classified as a, ~ and 7 [1,2]. Mitochondrial DNA polymerase is thought to be the 7 enzyme [3--6]. The physiological roles of these polymerases have not been clearly defined. The a enzyme has been implicated in DNA replication and the ~ enzyme in repair, but some reports are inconsistent [1,2,7--11]. Recent reports on aphidicolin, a tetracyclic diterpenoid obtained from Cephalosporium aphidicola Perch, have shown that aphidicolin inhibits DNA polymerase a but not the DNA polymerases ~ or 7 [12--17]. The specificity of aphidicolin for DNA polymerase a has been shown to be useful for enzymatic study of DNA synthesis [9--17].

414 The present communication concerns differences in the effect of aphidicolin on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse ascites sarcoma cells. Materials and Methods

Materials. The reagents used in these experiments were obtained from the following sources: [aH]dTTP, [aH]dGTP and [3H]dCTP from the Radiochemical Centre, deoxynucleoside triphosphates and 2',3'
415

control tubes for aphidicolin reactions. The reaction mixture (final volume: 0.6 ml) was incubated at 37°C for 10 min for replicative DNA synthesis and for 60 min for unscheduled DNA synthesis. Assays were performed in duplicate. The radioactivity incorporated into acid-insoluble materials was measured as described previously [19]. Results

Effect of aphidicolin on replicative DNA synthesis. We showed in previous studies that DNA synthesis in permeable cells was mostly replicative when supplied with the four deoxynucleoside triphosphates (dNTPs), ATP, Mg2÷ and the proper ionic environment [20]. Replicative DNA synthesis was inhibited by aphidicolin, as shown in Fig. 1. A resistant fraction to aphidicolin remained only 4% or le,~s of total activity. Mode of inhibition. To study the effect of the addition of one of dNTPs on aphidicolin-inhibition of replicative DNA synthesis, the concentration of all four dNTPs in the control assay system was reduced to 1 ~M each, and aphidicolin was added at 0.05 pg/ml (0.15 pM), a concentration high enough to inhibit about 70% of replicative DNA synthesis in the control system. The aphidicolin-inhibition was partially reversed by the addition of a high concentration of dCTP (500 /~M), but not by the other three dNTPs (Table I). The requirement of such a high concentration of dCTP to reverse the aphidicolininhibition suggested that aphidicolin was not a mere competitive inhibitor, with respect to dCTP, of replicative DNA synthesis in permeable SR-C3H/He cells. Lineweaver-Burk plots of replicative DNA synthesis in the presence of aphidicolin showed that aphidicolin was a mixed type inhibitor, affecting both apparent Km and V [24], of replicative DNA synthesis for dCTP (Fig. 2). The same mixed type of inhibition by aphidicolin with respect to dCTP was obTABLE I EFFECT OF AN ADDITIONAL AMOUNT OF ONE OF THE DEOXYNUCLEOSIDE TRIPHOSPHATES ON T H E I N H I B I T I O N O F R E P L I C A T I V E D N A S Y N T H E S I S B Y A ' P H I D I C O L I N R e p l l c a t i v e D N A s y n t h e s i s i n p e r m e a b l e S R - C 3 H / H e cells was m e a s u r e d as d e s c r i b e d i n M a t e r i a l s and M e t h o d s e x c e p t t h a t t h e c o n c e n t r a t i o n Of t h e all f o u r d N T P s w a s 1 ~M i n t h e c o n t r o l assay s y s t e m a n d t h a t s p e c i f i c a c t i v i t y o f t h e l a b e l e d c o m p o u n d ( [ 3 H ] d T T P or [ 3 H ] d G T P ) was 1.5 C i / m m o l . T h e c o n c e n t r a t i o n o f d N T P a d d e d t o t h e c o n t r o l assay s y s t e m was 5 0 0 ~M, E x p e r i m e n t s A a n d B w e r e c o n d u c t e d s e p a r a t e l y . A s s a y w a s p e r f o r m e d i n d u p l i c a t e . V a r i a t i o n i n t h e results f o r d u p l i c a t e t e s t s was less t h a n 12%. A c t i v i t y is e x p r e s s e d as c p m of [ 3 H ] d N M P i n c o r p o r a t e d / 1 0 7 cells p e r 10 rain.

Experimental system

[3H]dNTP

Activity a p h i d i c o l i n (/~g/ml) 0

0.05

Remaining activity (%)

A. C o n t r o l s y s t e m +dATP +dGTP +dCTP

[3H]dTTP [3H]dTTP [3H]dTTP [ 3 H] d T T P

7685 9330 7910 11 3 0 5

2120 2875 2110 4660

28 31 27 41

B. C o n t r o l s y s t e m +dATP +dTTP +dCTP

[3H]dGTP [ 3H]dGTP [3H]dGTP [ 3 H ] dGTP

7380 8033 7220 8133

2273 2100 1960 3420

31 26 27 42

416

2O

A6

%

\~ "~ 15

x

O I__ ~;~;

-0

~

~

0.2

,

,

1"

L

,

0.5 1 2 5 10 APHIDICOLI N (.ug/ml)

,

,

20

40

0

0:1

0:2 0:3 1/[dCZP] (pM)

0:4

Fig. 1. Effects of increasing c o n c e n t r a t i o n o f a p h i d i c o l i n o n replicative D N A s y n t h e s i s and u n s c h e d u l e d D N A s y n t h e s i s in p e r m e a b l e S R - C 3 H / H e cells. Replicative D N A s y n t h e s i s ( e ) w a s m e a s u r e d at 37°C for 10 rain in the assay m i x t u r e f o r D N A repHcass, and u n s c h e d u l e d D N A s y n t h e s i s (o) w a s m e a s u r e d at 37°C for 6 0 rain in the ATP-free assay m i x t u z e s u p p l e m e n t e d w i t h 0.22 mM b l e o m y c i n as described in Materials and M e t h o d s . A p h i d i c o l l n dissolved in d i m e t h y i s u l f o x i d e w a s a d d e d t o the assay m i x t u r e (0.6 ml in final v o l u m e ) at t h e i n d i c a t e d final c o n c e n t r a t i o n s in a v o l u m e of 0.01 ml. A n e q u a l v o l u m e of d t m e t h y l s u l f o x i d e w a s a d d e d to t h e c o n t r o l tubes. The activity is e x p r e s s e d as c pm of [3H]dTMP incorp o r a t e d / 1 0 7 cells per 1 0 rain for replicative D N A s y n t h e s i s and as c pm of [3H ]dTMP i n c o r p o r a t e d / 1 0 7 cells per 6 0 rain for u n s c h e d u l e d D N A synthesis. Fig. 2. Effect of dCTP c o n c e n t z a t t o n o n i n h i b i t i o n o f repllcative D N A s y n t h e s i s b y aphidicolin. Data are p l o t t e d a c c o r d i n g to L i n e w e a v e r and Burk. Repllcative D N A s y n t h e s i s w a s m e a s u r e d as described in Materials a n d M e t h o d s e x c e p t t h a t t h e specific activity of [3H]dTTP was 1.5 C i / m m o l and that t h e concentxation of dCTP was varied as indicated. T h e c o n c e n t r a t i o n o f a p h i d i c o l i n in t h e r e a c t i o n m i x t u r e was as follows. 0 (e), 0 . 1 / J g / m l (o) and 0.3/~g/ml (~).

served when [3H]dCTP was used as a labelled compound. Aphidicolin inhibited replicative DNA synthesis noncompetitively with respect to dTTP (Fig. 3). The non-competitive nature o f aphidicolin-inhibition for dATP and dGTP was also suggested by the result shown in Table I. The presence of plural binding sites for dNTPs in the catalytic region of DNA polymerase a, as suggested by Okura and Yoshida [25], is thought to explain the difference between the effects o f dCTP and of the other three dNTPs on the aphidicolin-inhibition of replicative DNA synthesis. Effect of aphidicolin on unscheduled DNA synthesis. Following Kaufman et al. [26], the term 'unscheduled' DNA synthesis is used in the present paper to indicate non-replicative DNA synthesis occurring in vitro in nuclei or in permeable cells. DNA synthesis measured in bleomycin-treated permeable cells with ATP-free assay mixture (bleomycin-induced DNA synthesis) was shown to be unscheduled [22]. DNA synthesis in nuclei isolated from adult rat liver was not dependent on ATP, but was induced by bleomycin or by bleomycin and ATP (Fig. 4) [22,23]. The DNA synthesis in adult rat liver nuclei was largely unscheduled [22,23,26]. Aphidicolin did not affect unscheduled DNA synthesis in bleomycin-treated permeable sarcoma cells or in either bleomycin-treated or bleomycin-untreated rat liver nuclei (Figs. 1 and 4). Comparison with other DNA synthesis inhibitors. Replicative DNA synthesis in permeable cells was highly sensitive to AraCTP, N~thylmaleimide and

417

t /

5/

g-

S

x

Ig 0.

~5

z '~'

0

0.2 0.4 0.6 0.8 1/[dTTP] (pM)

1.0

o---

--o

°o~--~ 2'.5

O

1~

zG

45

APHIDICOLIN ( p g l m l )

F i g . 3. E f f e c t o f d T T P c o n c e n t r a t i o n o n i n h i b i t i o n o f r e p l l c a t i v e D N A s y n t h e s i s b y a p h i d i c o l i n . D a t a a r e p l o t t e d a c c o r d i n g t o L i n e w e a v e r a n d B u r k . R e p l i c a t i v e D N A s y n t h e s i s w a s m e a s u r e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s e x c e p t t h a t t h e c o n c e n t r a t i o n o f [ 3 H ] d T T P ( 1 . 5 C i / m m o l ) w a s v a r i e d as i n d i c a t e d . A p h i d i c o l i n w a s a d d e d t o t h e r e a c t i o n m i x t u r e a t t h e c o n c e n t r a t i o n o f 0 ( e ) , 0 . 1 5 /~g/ml (o) a n d 0 . 5

pglml (A). Fig. 4 . E f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n o f a p h i d i c o l l n o n u n s c h e d u l e d D N A s y n t h e s i s i n i s o l a t e d r a t liver n u c l e i . D N A s y n t h e s i s i n r a t l i v e r n u c l e i w a s m e a s u r e d a t 3 7 ° C f o r 6 0 m i n i n t h e a b s e n c e o f b l e o m y c i n a n d A T P ( o ) , in t h e p r e s e n c e o f 2 . 5 m M A T P ( e ) , in t h e p r e s e n c e o f 0 . 2 2 r a M b l e o m y c i n (~), o r in t h e p r e s e n c e o f b l e o m y c i n a n d A T P (&). A p h i d i c o l i n d i s s o l v e d i n d i m e t h y i s u l f o x i d e w a s a d d e d t o t h e assay mixture at the indicated final concentrations in a volume of 0.01 mi. Control tubes contained the s a m e c o n c e n t r a t i o n o f d i m e t h y l s u l f o x i d e . T h e a c t i v i t y is e x p r e s s e d as c p m o f [ 3 H ] d T M P i n c o r p o r a t e d / 107 nuclei per 60 rain.

T A B L E II DIFFERENTIAL EFFECTS OF SOME INHIBITORS SCHEDULED DNA SYNTHESIS

ON REPLICATIVE

DNA SYNTHESIS AND UN-

P e r m e a b l e cells a n d r a t liver n u c l e i w e r e p r e p a r e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s e x c e p t t h a t 2 - m e x c a p t o e t h a n o l w a s o m i t t e d f r o m t h e b u f f e r t o p r e p a r e p e r m e a b l e cells o r n u c l e i t o t e s t t h e e f f e c t o f N-ethyhnaleimide. The inhibitor was added to the assay mixture at the indicated final concentration. Repllcative DNA synthesis was measured at 37°C for 10 rain in the assay mixture for DNA replicase. Un, s c h e d u l e d D N A s y n t h e s i s i n p e r m e a b l e cells w a s m e a s u r e d i n t h e A T P - f r e e a s s a y m i x t u r e s u p p l e m e n t e d w i t h 0 . 2 2 m M b l e o m y e i n a t 3 7 ° C f o r 6 0 r a i n as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . U n s c h e d u l e d D N A s y n t h e s i s in r a t liver n u c l e i w a s m e a s u r e d i n A T P - f r e e a s s a y m i x t u r e . P e r c e n t i n h i b i t i o n w a s c a l c u l a t e d relative to the activity measured in the absence of any inhibitors. Each value represents the average of two determinations. Inhibitor

% Inhibition of Replicative DNA synthesis

Unscheduled DNA synthesis P e r m e a b l e cells

R a t liver n u c l e i

0 23 0 0 84

27 17 0 15 86

P e r m e a b l e ceHs AraCTP (100 ~M) N - E t h y l m a l e i m i d e (1 m M ) Aphidicolin (1/~g/ml) E t h i d i u m b r o m i d e ( 2 0 ~M) 2',3'-ddTTP (10/~M)

96 100 96 89 21

418 ethidium bromide, whereas unscheduled DNA synthesis measured in permeable cells or in rat liver nuclei was less sensitive to these drugs (Table II). Unscheduled DNA synthesis was also inhibited by increasing the concentration of these drugs. Aphidicolin was without effect on unscheduled DNA synthesis even at the high concentration of 40 ~g per ml of reaction mixture. Unscheduled DNA synthesis was more sensitive to 2',3'
In the present communication, DNA polymerases engaged in replicative DNA synthesis in permeable SR-C3H/He cells and in unscheduled DNA synthesis in permeable cells or rat liver nuclei were studied by using aphidicolin, which is known to be a selective inhibitor of DNA polymerase a [12--17]. The present results confirm the inhibitory effect of aphidicolin on replicative DNA synthesis [9--11,13--17]. Aphidicolin inhibited selectively and almost completely replicative DNA synthesis in permeable SR-C3H/He cells, suggesting that DNA polymerase a was the main polymerase engaged in replicative DNA synthesis. The aphidicolin-inhibition of replicative DNA synthesis was mixed competitive and noncompetitive (mixed type) inhibition with respect to dCTP and noncompetitive with respect to the other three dNTPs. Oguro et al. [28] reported that aphidicolin inhibited DNA polymerase a by competing with dCTP and not by competing with the other three dNTPs. They also reported that DNA synthesis in isolated sera urchin nuclei and in isolated HeLa nuclei was inhibited by aphidicolin non~ompetitively with respect to any of the four dNTPs [29]. Further studies are required to clarify the reason why there are differences in these preparations in the inhibition modes by aphidicolin. However, considering that DNA polymerase a is thought to be the major ~olymerase engaged in replicative DNA synthesis and, so far, DNA polymerase a is the sole enzyme sensitive to aphidicolin, the differences in the inhibition modes by aphidicolin may be due to the presence of conformational differences in DNA polymerase a between purified DNA polymerase a-template system and DNA replication complexes. Unscheduled DNA synthesis in bleomycin-treated permeable cells and isolated rat liver nuclei was completely resistant to aphidicolin. Compared with replicative DNA synthesis, unscheduled DNA synthesis was markedly resistant to N,ethylmaleimide and AraCTP, and was highly sensitive to 2',3'
419

DNA synthesis showed different results from ours. The repair modes of DNA synthesis in nuclei isolated from ultraviolet-treated HeLa cells [9], in human lymphocytes permeabilized after ultraviolet-irradiation [10] and in lysed human fibroblasts pretreated with ultraviolet light or chemical carcinogens [11] were shown to be highly sensitive to aphidicolin. These studies [9--11] suggested that the major DNA polymerase engaged in repair modes of DNA synthesis was DNA polymerase a. A possible explanation on the above inconsistent results on repair DNA polymerase is that DNA polymerase involved in repair (unscheduled) DNA synthesis is not identical for all of the kinds of DNA damage that undergoes repair. Species differences (e.g. between primates and rodents) in repair enzymes may also be considered. To clarify this, we are trying to study repair enzymology in various preparations. Acknowledgements The authors wish to thank Dr. S. Urano (Radioisotope Laboratory), Dr. T. Taguchi (Biochemical Laboratory) and Mr. S. Nakano (Radioisotope Laboratory) of Tokyo Metropolitan Institute of Gerontology for their cooperation in the preparation of aphidicolin. We are also indebted to Nippon Kayaku Co. for providing copper-free bleomycin A2. This investigation was supported in part by a grant from the Japan Ministry of Education, Science and Culture. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Weissbach, A. (1977) Ann. Rev. Biochem. 46, 25--47 Shetnin, R., Hu mb ert, J. and Pearlman, R.E. (1978) Ann. Rev. Biochem. 4 7 , 2 7 7 - - 3 1 6 Bolden, A., Noy, G.P. and Weissbach, A. (1977) J. Biol. Chem. 252, 3 3 5 1 - - 3 3 5 6 Bertazzoni, U., Scovassi, A J . and Brun, G.M. (1977) EULr. J. Biochem. 8 1 , 2 3 7 - - 2 4 8 Hiibscher, U., Kuenzle, C.C. and Spadari, S. (1977) Ettr. J. Biochem. 8 1 , 2 4 9 - - 2 5 8 Tanaka, S. and Koike, K. (1978) Biochem. Biophys. Res. C o m m u n . 8 1 , 7 9 1 - - 7 9 7 Butt, T.R., Wood, W.M. and Adams, R.L.P. (1976) Biochem. Soc. Trans. 4 , 8 0 7 - - 8 1 0 Butt, T.R., Wood, W.M., McKay, E.L. and Adams, R.L.P. (1978) Biochern. J. 1 7 3 , 3 0 9 - - 3 1 4 Hanaoka, F., Kato, H., Ikegami, S., Ohashi, M. and Yamada, M. (1979) Biochem. Biophys. Res. C o m m u n . 87,575---580 Berger, N.A., Kuxohara, K.K., Petzold, S.J. and Sikorski, G.W. (1979) Biochem. Biophys. Res. Commun. 89, 218--225 Ciarrocchi, G., Jose, J.G. and Linn, S. (1979) Nucleic Acid Res. 7, 1205--1219 Ohashi, M., Taguchi, T. and Ikegami, S. (1978) Biochem. Biophys. Res. C o m m u n . 82, 1 0 8 4 - - 1 0 9 0 Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H. and Mano, Y. (1978) Nature 2 7 5 , 4 5 8 - 460 Wist, E. and Prydz, H. (1979) Nucleic Acid Res. 6, 1 5 8 3 - - 1 5 9 0 Noy , G.P. and Spadari, S. (1979) Biochem. Biophys. Res. C o m m u n . 88, 1 1 9 4 - - 1 2 0 2 Longiaru, M., Ikeda, J., Jarkovsky, Z., Horwitz, S.B. and Horwitz, M.S. (1979) Nucleic Acid Res. 6, 3369--3386 Krokan, H., Schaffer, P. and DePamphilis, M.L. (1979) Biochemistry 18, 4 4 3 1 - - 4 4 4 3 Dalziel, W., Hesp, B., Stevenson, K.M. and Jarvis, J.A.J. (1973) J. Chem. Soc., Perkin Trans. 1, 2 8 4 1 - 2851 Seki, S. and Oda, T. (1977) Cancer Res. 3 7 , 1 3 7 - - 1 4 4 Seki, S. and Oda, T. (1977) Biochim. Biophys. Acta 476, 24--31 Seki, S., Watanabe, S. and Oda, T. (1980) Biochim. Biophys. Acta 607, 36--42 Seki, S. and Oda, T. (1978) Biochim. Biophys. Acta 5 2 1 , 5 2 0 - - 5 2 8 SekJ, S. and Oda, T. (1977) Biochirn. Biophys. Acta 479, 391--399 Dixon, M. and Webb, E.C. (1969) Enzymes, 3rd edn. pp. 332--352, L o n g m a n Group Ltd., L o n d o n Okttra, A. a n d Yoshida, S. (1978) J. Biochem. ( T o k y o ) 8 4 , 7 2 7 - - 7 3 2 Kaufman, D.G., Grisham, J.W. and S t e n s t r o m , M.L. (1972) Biochim. Biophys. A c t a 272, 212--219

420 27 Seki, S. and Oda, T. (1980) Biochim. Biophys. Acta 6 0 6 , 2 4 6 - - 2 5 0 28 Oguro, M., Suzuki-Horl, C., Nagano, H., Mano, Y. and Ikegami, S. (1979) Eur. J. Biochem. 9 7 , 6 0 3 - 607 29 Oguro, M., Shloda, M., Nagano, H., Mano, Y.0 Hanaoka, F. and Yamada, M. (1980) Biochem. Biophys. Res. Commun. 92, 13--19 30 Waqaz, M.A., Evans, M.J. and Huberman, J.A. (1978) Nucleic Acid Res. 5, 1933--1946 31 Hiibscher, U., Kuenzle, C.C. and Spadari, S. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2316--2320