Mode of action of 9-β-d -arabinosyladenine and 1-β-d -arabinosylcytosine on DNA synthesis in human lymphoblasts

57

Biochimica et Biophysica Acta, 606 (1980) 57--66 © Elsevier/North-Holland Biomedical Press

BBA 99591

MODE OF ACTION OF 9-~-D-ARABINOSYLADENINE AND 1-~-D-ARABINOSYLCYTOSINE ON DNA SYNTHESIS IN HUMAN LYMPHOBLASTS

DOUGLAS E. BELL and ARNOLD FRIDLAND

Division of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, P.O. Box 318, Memphis, TN 38101 (U.S.A.) (Received June 21st, 1979)

Key words: DNA synthesis; Arabinosyladenine; Arabinosylcytosine; (Human lymphoblast)

Summary The effects of 9-~-D-arabinosyladenine (AraAde), 1-~-D-arabinosylcytosine (AraCyt) and 2'-deoxyadenosine on DNA replication in cultured human lymphoblasts (CCRF-CEM line) were studied by pulse-labeling cells with [3H]thymidine and analyzing the nascent DNA by velocity sedimentation in alkaline sucrose gradients. At doses that reduced the overall rate of DNA synthesis to 50--70% of control values, both AraAde and AraCyt profoundly inhibited the formation of new replicons, with secondary effects on chain elongation contributing to the total inhibition of DNA synthesis. In contrast, the suppression of DNA synthesis by 2'-deoxyadenosine stemmed mainly from an inhibition of chain elongation. These studies also disclosed that about 100 times more AraAde than AraCyt was required to produce a similar inhibition of DNA replication in CCRF-CEM cells. Determination of intracellular concentrations of the nucleoside triphosphates (AraCTP and AraATP) indicated that 90% inhibition of DNA synthesis was achieved at 1.6 and 25 pmol/1.106 cells, respectively. Studies with cell lysates revealed that the replicative machinery in CCRF-CEM cells is more sensitive to AraCTP than to AraATP. This finding contrasts with earlier research, in which the inhibition of purified DNA polymerase by either AraATP of AraCTP yielded essentially the same Ki value. The difference in sensitivity of the cell lysate to these arabinonucleotides may reflect either a target enzyme other than DNA polymerase or, more plausibly, some subtle interaction of the polymerase with other components of the replicative process.

Abbreviation: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; AraAde, 9-~-D-a~abinosyl-

adenine; AraCYt, 1-~-D-arabinosylcytosine.

58 Introduction Nucleoside antibiotics such as 9-fl-D-arabinosyladenine (AraAde) and 1-/3-Darabinosylcytosine (AraCyt) have become increasingly important as antitumor and antiviral agents [ 1]. AraCyt, in particular, has been shown effective against acute granulocyte leukemia [2,3], while AraAde has produced encouraging results in the treatment of certain viral diseases [4]. An important route for metabolic activation of these c o m p o u n d s in mammalian cells is their conversion to 5'-triphosphates (either AraATP or AraCTP), and their c y t o t o x i c effects have been ascribed to these nucleoside derivatives [1]. Although studied intensively, the mode of action of the arabinonucleosides is still uncertain. As triphosphates, the c o m p o u n d s can inhibit ribonucleoside diphosphate reductase [5,6]; AraATP and AraCTP are also potent inhibitors of some DNA polymerases and their incorporation into nascent DNA may block or slow the replication of DNA [7--10]. Recent work from this laboratory has disclosed a new mode of action for AraCyt, namely, inhibition of replicon initiation, which apparently is distinct from the c o m p o u n d ' s effect on chain elongation [11,12]. Described here are experiments in which we determined the inhibitory effect of AraAde, AraCyt and 2'-deoxyadenosine (dAdo) on initiation and elongation of human lymphoblast DNA. The results show that both AraAde and AraCyt, b u t n o t dAdo, inhibit the formation of new replicons, with higher concentrations of AraAde required for an inhibitory effect. This effect of AraAde and AraCyt on replicon synthesis could not be explained from an inhibition of DNA chain polymerization but more likely resulted from differences in the degree of interaction of the arabinose-containing nucleosides with the different proteins required for DNA replication. Furthermore, using a cell lysate of human lymphoblasts, we show that the replicative machinery in CCRF-CEM cells responds differently to the triphosphates of AraAde and A r a C y t . Materials and Methods

Cells. Human lymphoblasts (CCRF-CEM line) were cultured in stationary suspensions, as described earlier [ 11 ]. Chemicals. 2'-Deoxyadenosine was purchased from Sigma Chemical Company; 2-deoxycoformycin was generously provided by Dr. John D. Douros, Developmental Therapeutics Program, Chemotherapy, National Cancer Institute, and AraAde was provided by Dr. Harry B. Wood, Jr., Drug Synthesis and Chemistry Branch, Division of Treatment, National Cancer Institute. AraCyt was obtained from the Upjohn Company, while araATP and AraCTP were purchased from PL-Biochemicals. Sedimentation analyses in sucrose gradients. The DNA of pulse-labeled cells was analyzed by sucrose gradient centrifugation as follows. Cells were incubated at 37°C in the presence of inhibitors at a cell concentration of a b o u t 5 • l 0 s cells/ml. Immediately before the addition of drug and at 15, 30 and 60 min later, 2-ml aliquots were removed and added to 5 pCi of [methyl-3H]dThd (20 Ci/mmol) and pulse-labeled for 5 min. The pulse was terminated by adding the cells to 12 ml of ice-cold medium containing 0.9% NaC1, 0.01 M potassium

59 phosphate, 0.01 M KCN, and 0.008 M EDTA (phosphate-buffered saline/KCN/ EDTA) followed by centrifugation for 10 min at 700 × g in an International centrifuge, model PR-2, operated at 4°C. The pelleted cells were resuspended in phosphate-buffered saline/KCN/EDTA medium and layered on 0.5 ml of lysing solution on top of a 36 ml, 5--20% alkaline sucrose gradient containing 0.8 M NaC1, 0.25 M NaOH, and 0.01 M EDTA. The lysing solution contained 0.2 M NaOH, 0.02 M EDTA, and 0.1% Nonidet P-40. The cells were lysed overnight at 4°C in the dark. The gradients were centrifuged at 20 000 rev./min for 5 h at 4°C in a Beckman SW27 rotor and fractionated with an ISCO model 640 fractionator. 100 /~g of salmon-sperm DNA (Sigma) were added to each fraction before precipitation by addition of cold trichloroacetic acid. Precipitates were collected on Whatman G F / A filter discs, washed three times with 5% trichloroacetic acid/l% sodium pyrophosphate and dried. Radioactivity was determined by liquid scintillation spectrometry with a Searle Mark III scintillation counter. DNA pulse-labeled in vitro with [ 3H]TTP was analyzed by velocity sedimentation in alkaline sucrose gradients. At various times after addition of [3H]TTP, 0.2-ml aliquots of lysate suspension were removed and layered on 0.5 ml lysing solution on top of a 36-ml, 15--30% alkaline sucrose gradient. After incubation at 37°C for 4 h, the gradients were centrifuged for 4 h at 26 000 rev./min in a SW-27 rotor at 15°C. Gradients were fractionated as described above. Isolation o f AraAde and AraCyt nucleotides. To determine the intracellular concentrations of AraAde and AraCyt metabolites, we incubated cells in media containing either 0.05 pM [5,6-3H]AraCyt (13.2 Ci/mmol) or 5 uM [2,8-3H] AraAde (1.45 Ci/mmol) (New England Nuclear). At different times after the addition of radioactive drugs, 5-ml aliquots of cells were removed and added to 25 ml of ice-cold phosphate-buffered saline/KCN/EDTA. The cells were centrifuged for 10 min at 700 × g at 4°C. Cells were washed once with phosphate-buffered saline/KCN/EDTA, and the washed pellet was resuspended in 0.3 ml 5% trichloroacetic acid. The cell suspension in trichloroacetic acid was kept on ice for 10 min and then centrifuged for 10 min at 700 × g. The supernatant fluid was decanted and extracted five times each with 1 ml of watersaturated diethyl ether. The aqueous layer was lyophilized and the powder was redissolved in 0.03 ml of water. Aliquots of the cell extract were spotted on polyethyleneimine-cellulose thin-layer chromatography plates and 5 pl of a marker solution containing dCyd, dCMP, dCDP, dCTP (for AraCyt analysis) or dAdo, dAMP, dADP, dATP (for AraAde analysis) were applied to each cell extract spot. The plates were developed first in 1 M acetic acid at 4°C until the solvent front was about 2 cm above the origin. They were then transferred without drying to a tank containing 1 M acetic acid and 3 M LiC1 (2 : 1, v/v), and development was continued at 4°C until the solvent front was a b o u t 15 cm above the origin. Each plate was dried and ultraviolet-absorbing spots were marked, cut o u t and scraped into scintillation vials. Nucleotides were extracted from the polyethyleneimine-cellulose with 1.5 ml of 1.5 M NaC1 before 14 ml ACS (Amersham Corporation) were added to each vial for liquid scintillation counting. DNA synthesis in lysates. In vitro assays of the synthesis of D N A b y lympho-

60 blasts were done with cells lysed by Nonidet P-40 treatment. Log-phase cells were centrifuged and resuspended to a concentration of 3 . 1 0 7 cells/ml in a buffer containing 20 mM Hepes, pH 7.6, 130 mM KC1, 10 mM MgC12, 2 mM dithiothreitol, 10% glycerol (v/v), and 4% dextran (w/v); Nonidet P-40 was added to give a final concentration of 0.1%. The cells were incubated for 5 min at 0°C with Nonidet P-40 and observed under phase-contrast microscopy. Under these conditions, at least 99% of the cells were lysed with no detectable damage to the nuclei. To assay DNA synthesis, 1.0 ml of the lysate was added to 3.0 ml of the same buffer, which also contained 2.5 mM phosphoenolpyruvate, 2.5 mM ATP, and 50 pM of dGTP, dATP and dCTP. dCTP was omitted in experiments with AraCTP and dATP was not included in the AraATP experiments. 5 ~M [3H]TTP (1.5 Ci/mmol) was also included in the reaction mixture. The incorporation of [3H]TTP into acid-precipitable material was determined by removing 0.1 ml aliquots at various times and adding these to 1.5 ml of 0.4 M NaOH containing 0.5 mg carrier DNA. The samples were incubated at room temperature in the NaOH for 3 min before the addition of 1.0 ml of ice-cold 10% trichloroacetic acid/6% sodium pyrophosphate. After remaining on ice for a few minutes, the trichloroacetic acid precipitates were collected on Whatman G F / A filter discs and processed as described for gradient fractions. Results Fig. 1 illustrates the effect of each agent on the incorporation of radioactive thymidine into acid-precipitable material of CCRF-CEM cells. In experiments with AraAde and dAdo, 1.8 ~M 2'-deoxycoformycin was included in the incubation media to prevent deamination of these adenine-containing derivatives [13]. 2'-Deoxycoformycin at this concentration had no effect on either the

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30 60 90 120 Time Afler Addilion of Drug (rain) Fig. 1. I n h i b i t i o n o f D N A s y n t h e s i s b y A r a A d e , AraCYt, and d A d o . Cells w e r e i n c u b a t e d w i t h e i t h e r

5 ~M A.raAde (m), 2 5 /~M 2 ' - d e o x y a d e n o s i n e (A), o r 0 . 0 5 ~ M A r a C y t (e). Cells i n c u b a t e d w i t h d A d o also w e r e p r e i n c u b a t e d f o r 3 0 m i n w i t h 1 . 8 / z M 2 ' - d e o x y c o f o r m y c i n b e f o r e t h e a d d i t i o n o r d A d o . A t t h e various t i m e s during the i n c u b a t i o n w i t h d r u g , a l i q u o t s o f cells w e r e r e m o v e d t o 0 . 5 /JCi [ 3 H ] d T h d and p u l s e - l a b e l e d f o r 5 m i n . The p e r c e n t i n h i b i t i o n o f D N A s y n t h e s i s m i n e d b y c o m p a r i n g t h e i n c o r p o r a t i o n o f [ 3 H ] d T h d in d r u g - t r e a t e d cells vs. c o n t r o l cells.

AraAde or of AraAde and added w a s deter-

61 synthesis of DNA or the growth of CCRF-CEM cells. The results show that 5 uM AraAde inhibited DNA synthesis by about 50% within 30 min, while 25 pM dAdo was required to produce a similar inhibition. The most potent inhibitor of DNA was AraCyt: only 0.05 pM of the compound was required to produce 70% inhibition within 30 min. The effect of the compounds on replicon initiation and elongation of DNA precursor strands was examined by pulse-labeling cells with [3H]Thd at various times after the addition of inhibitor and analyzing the nascent DNA in alkaline sucrose gradients. As shown by the gradient profiles in Fig. 2, all three agents sharply inhibited the replication of DNA in CCRF-CEM cells but with different specificities. Treatment of cells for increasing intervals with AraAde and AraCyt before labeling with [3H]dThd caused a progressive shift of the profiles of radioactivity toward the high molecular weight DNA (Fig. 2B and C). By contrast, dAdo uniformly inhibited the labeling of molecules of every size, as shown by the similarity of gradient profiles at different times (Fig. 2A). This preferential suppression of labeling at low molecular weight DNA (fractions 2--8) most probably stemmed from an inhibition of replicon initiation by AraAde and AraCyt [12]. That chain elongation was also inhibited by these drug concentrations is reflected by the lower amounts of radioactivity incorporated into the larger molecular weight DNA (fractions 9--20). The only effect obviously produced by dAdo was a suppression of chain elongation. The different modes of action by the arabinonucleosides vs. dAdo suggests that replicon initiation and chain elongation can be inhibited independently. The preceding experiments demonstrated that about 100 times more AraAde than AraCyt is required to inhibit DNA synthesis in CCRF-CEM cells (Fig. 1). This could stem either from differences in the metabolism of the two drugs or from a difference in sensitivity of the DNA replication process to the active

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Fig. 2. V e l o c i t y s e d i m e n t a t i o n profiles o f D N A o f drug-treated cells pulse-labeled w i t h [ 3 H ] d T h d . Cells w e r e pulse-labeled and the D N A w a s a n a l y z 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 ) at v a r i o u s t i m e s a f t e r t h e drug addition: • i m m e d i a t e l y b e f o r e a d d i t i o n ; o, 1 5 r a i n a f t e r ; ~, 3 0 r n i n ; e, 6 0 r a i n after. Cells t r e a t e d w i t h e i t h e r d A d o o r A r a A d e w e r e p r e i n c u b a t e d w i t h 1 . 8 / ~ M 2 ' - d e o x y c o f o r r n y c i n for 3 0 r a i n . T h e D N A p r o f i l e s r e p r e s e n t cells t x e a t e d w i t h ( A ) 2 5 / J M d e o x y a d e n o s i n e ; (B) 5 . 0 / ~ M A r a A d e o r (C) 0 . 0 5 # M A r a C y t . T h e d i r e c t i o n o f s e d i m e n t a t i o n is t o the r i g h t .

62 forms of AraAde and AraCyt. To distinguish between these possibilities, we studied the cellular metabolism of radioactively labeled AraAde and AraCyt. Exponentially growing cells were incubated with either 0.05 ~M [ 3H] AraCyt or 5 ~M [3H]AraAde and, at various intervals, the radioactivity incorporated into the various nucleotides was determined by thin-layer chromatography. Consistent with previous studies [14,15], 5'-triphosphates were the predominant intracellular forms of AraAde and AraCyt identified (Table I). At 5 pM exogenous AraAde, the average maximum concentration of AraATP was 25.4 pmol/1 • 106 cells, while 0.05 pM AraCyt resulted in a maximum concentration of a b o u t 1.6 pmol of AraCTP/1 • 106 cells; thus, a 100-fold greater concentration of AraAde than of AraCyt caused the formation of 16 times more AraATP than AraCTP, at a time when steady state had been achieved in the lymphoblasts. To determine the effect of AraATP and AraCTP on the process of replication, we examined DNA synthesis in vitro using lysates prepared from CCRFCEM cells. As reported by other groups [16,17], the lysate catalyzed the incorporation of [ 3H] dTTP into an acid-precipitable fraction, dependent on the presence of ATP (Fig. 3). When either ATP or deoxynucleoside triphosphates were omitted, the incorporation was diminished by 85 and 70%, respectively (data not shown). The residual activity in the absence of ATP or deoxynucleoside triphosphates can probably be attributed to residual nucleotide pools in the lysate. Obtaining maximal activity in this system required the addition of 4% dextran. Similar results have been reported for lysates of mouse cells [17] and homogenates of Physarum polycephalum [18]. Although the mechanism by which dextran stimulates DNA synthesis in vitro has n o t been further investigated, in this system the polysaccharide prevented clumping of the lysed cells during incubation. In mammalian cells, semiconservative DNA replication involves the formation of various intermediates. In alkaline sucrose gradients, most of these intermediates sediment at rates below 100 S, while preexisting bulk DNA sediments between 150 and 200 S [19,20]. To test whether DNA replication was similar in the lysate, we prelabeled CCRF-CEM cells with [14C]dThd, lysed them with Nonidet P-40 and pulse-labeled in vitro with [ 3H]dTTP. After different incuba-

TABLE I INTRACELLULAR AMOUNTS OF AraAde AND AraCyt NUCLEOTIDES FOUND AFTER VARIOUS TIMES OF I N C U B A T I O N OF CCRF-CEM CELLS WITH [ 3 H ] A r a A d e OR [ 3 H ] A r a C y t D e t a i l s o f a n a l y s i s axe p r e s e n t e d i n M a t e r i a l s a n d M e t h o d s . A r a - C M P l e v e l s w e r e f o u n d t o b e l e s s t h a n 10% of t h e t o t a l r a d i o a c t i v i t y a n d are n o t r e p o r t e d h e r e . n . d . , n o t d o n e .

Time after drug addition (rain)

Nucleotide

AraCTP

AraC DP

AraATP

A r a A DP

A r a A MP

10 30 60 90 120

0.13 0.42 1.43 1.59 n.d.

0.03 0.07 0.10 0.12 n.d.

14.35 22.95 23.75 31.96 22.95

10.35 15.17 20.20 20.00 15.65

4.70 6.60 8.80 7.5 6.17

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Fig. 3. E f f e c t o f A T P o n D N A s y n t h e s i s b y cell lysates. T h e rate o f s y n t h e s i s was d e t e r m i n e d as d e s c r i b e d in Materials a n d M e t h o d s , e i t h e r w i t h (o) o r w i t h o u t ( i ) A T P a n d phosphoenolpyruvate. Fig. 4. V e l o c i t y s e d i m e n t a t i o n analysis o f l y s a t e D N A . Whole cells w e r e p r e i n c u b a t e d o v e r n i g h t w i t h [ 1 4 C ] T h d ( 0 . 0 2 Ci]l, 4 5 C i / m o l ) t o l a b e l t h e b u l k D N A . T h e s e cells w e r e t h e n l y s e d a n d i n c u b a t e d w i t h [ 3 H ] T T P as d e s c r i b e d in Materials a n d M e t h o d s . A l i q u o t s o f t h e l y s a t e w e r e r e m o v e d at 5 rain (u), 10 vain (A) a n d 20 vain (o) a n d a n a l y z e d as d e s c r i b e d . ©, [ 1 4 C ] D N A .

tion times, the size distributions of ['4C]DNA and [3H]DNA were analyzed in alkaline sucrose gradients. The radioactivity profiles of bulk [ 14C]DNA (Fig. 4) from smnples taken from either intact cells or lysed cells were superimposable and sedimented with a median value of about 160 S. Thus, preexisting bulk DNA was not measurably degraded during lysis or during incubation of the lysate. The radioactivity profiles of [3H]DNA after a 5-min pulse showed a heterogeneous size distribution of nascent chains with a peak at about 24 S (Fig. 4). During longer pulses of 10 and 20 min, the radioactivity was incorporated into progressively longer DNA molecules (Fig. 4). After about 30 min, however, the incorporation of labeling into DNA stopped. Similar profiles were also obtained after cells were pulsed for 10 min (median at about 32 S) and chased for 30 and 60 min (data not shown). Thus, the size distribution of growing chains in vitro was remarkably similar to that of DNA chains pulse-labeled in vivo, except that, in vitro, nascent strands were not converted to bulk DNA. When either AraATP or AraCTP was added to this lysed cell preparation, the synthesis of DNA was inhibited, as evidenced by the decreased rates of [3H]dTTP incorporation into the trichloroacetic acid precipitate (data not shown). Fig. 5 depicts the inhibitory effect of AraCTP and AraATP on the activity of the complete lysate system. Inhibition was apparent at a drug concentration of about 0.1 pM and 1 gM for AraCTP and AraATP, respectively, and increased with increasing drug concentrations. The most striking feature of the data in Fig. 5 is that the effective concentration of AraCTP for 50% inhibition is about

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2].0 410 60 8.0 I0.0 Concentration of Arobinosyl Triphosphate (u,M) Fig. 5. T h e e f f e c t o f A r a C T P a n d A r a A T P o n t h e i n c o r p o r a t i o n o f [ 3 H ] T T P i n t o D N A o f cells l y s e d w i t h N o n i d e t P-40. A f t e r cell lysis, [ 3 H ] T T P i n c o r p o r a t i o n was m e a s u r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e r a t e s o f D N A s y n t h e s i s in t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f A r a C T P (4) or A r a A T P ( e ) a r e p l o t t e d as a p e r c e n t a g e o f t h e r a t e in t h e a b s e n c e o f a d d e d d r u g s .

15 times lower than for AraATP. Thus, the differential effect of the two drugs on D N A synthesis observed in intact cells could also be produced in the lysate system. This difference in sensitivity is apparently not due to a difference in competition by the intracellular pools of dCTP and dATP, because these compounds have been found in essentially equal concentrations in these cells [21]. In addition, determination of the fate of AraATP and AraCTP in lysate preparation disclosed that the two triphosphates were not degraded during the experiments {Table II). Thus, the higher activity of AraCTP as compared to AraATP is not the result of a preferential degradation of the latter nucleotide in the lysate.

TABLE

If

STABILITY

OF ARABINOSYL

TRIPHOSPHATES

IN C E L L

LYSATES

Cells were incubated for 1 h with either 5 # M [ 3 H ] A r a A d e or 0.1 /~M [3H]AraCyt. After this time, the cells were washed, lysed and added to the complete D N A synthesis solution in vitro as described in Materials and Methods. At the indicated time, aliquots of the lysed cell suspension were r e m o v e d and analyzed for A r a A T P or A r a C T P as described for Table I. T i m e a f t e r lysis ( r a i n )

Nucleotide concentration (pmol/1 • 10 6 cells) AraATP

AraCTP

0 10

29.3 31.2

3.5 3.5

20

28.8

2.7

30

22.8

3.1

65

Discussion As might be expected from their structural similarity, AraAde and AraCyt produce comparable effects on DNA synthesis. Their inhibitory activity appears to involve t w o distinct steps: a block of the formation of new replicons followed by inhibition of DNA chain propagation. Despite these findings, the molecular basis of AraCyt and AraAde action on DNA synthesis remains uncertain. Both agents function as reversible inhibitors o f mammalian DNA polymerases, but such inhibition would not be expected to affect replicon initiation [19]. One explanation for the dual effect of AraAde and AraCyt on DNA replication is that, in vivo, the initiation of new replicons is repressed when replication is inhibited. This possibility is unlikely, however, since with dAdo there is no apparent inhibition of replicon formation despite a strong inhibition of DNA synthesis. A second possibility is that the arabinonucleosides exert a preferential effect on a target involved in DNA chain initiation. Recently, Bjursell et al. have shown that 2'-azidocytidine, which differs from AraCyt only in substitution of an N3 group for an OH in the 2'-position, interferes primarily with initiation at the origin of replication of p o l y o m a DNA [22]. Reichard et al. [23], moreover, have obtained evidence with Escherichia coli that DNA primase, an enzyme required for DNA chain initiation, is the primary target for this drug. Thus, it is tempting to speculate that the effect of AraAde and AraCyt on replicon formation is due to a specific inhibition of an enzyme involved in initiation of replicon formation in mammalian cells. A third possibility is that the incorporation of these nucleoside analogs into nascent DNA s o m e h o w causes a preferential termination of synthesis at its origin. DNA synthesis in lysates prepared from CCRF-CEM cells by treatment with Nonidet P-40 was similar to that observed in other in vitro systems, in that incorporation of [3H]dTTP required all four deoxyribonucleoside triphosphates, ATP and MgC12 for maximum activity [16,17]. ATP is required in prokaryotic DNA replication in vitro to allow the formation of an initiation complex [24]. If eukaryotic replication is analogous, the strong ATP requirement (Fig. 3) suggests that the in vitro system described here is indeed incorporating radioactivity primarily by a replicative process. In accord with earlier reports [8,25,26], AraCTP and AraATP sharply inhibited DNA synthesis in vitro. However, the drugs differed markedly in their inhibitory activity: AraCTP was at least 15-fold more active in inhibition of DNA replication, both in vitro and in vivo. This contrasts with studies in which the agents inhibited purified DNA polymerase with about the same efficiency [27,28]. At present, we do n o t know whether this is simply an indication of a stronger interaction of AraCTP with the same target or whether additional molecular activities are involved. Further work with this lysate system will be required for more detailed information.

Acknowledgments We thank Drs. Arnold Welch and Thomas P. Brent for helpful discussions, John Gilbert for reading the manuscript and Ms. Jane Brown for skilled

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