Inhibition of Mammalian DNA Polymerase by the 5'-Triphosphate of 9-Jo-D-Arabinofuranosyladenine1

Home , Adenosine, Cyclic nucleotide, Cytosine, Deoxyguanosine, Nucleoside triphosphate

[CANCER RESEARCH 27, 1528-1533, September 1967] Inhibition of Mammalian DNA Polymerase by the 5'-Triphosphate of 9-jo-D-Arabinofuranosyladenine1

J. J. FORTH-' AND SEYMOUR S. COHEN

Department of Pathology and the Department of Therapeutic Research, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

SUMMARY polymerization by polynucleotide phosphorylase of E. coli of The 5'-triphosphate of 9-0-D-arabinoturanosyladenine (ara- ADP and CDP to polyadenylate and polycytidylate, respectively, were sharply inhibited by ara-CDP (2) and by ara-ADP (12), ATP) was tested as a substrate or inhibitor of polynucleotide reactions leading to polymerization by this enzyme are not synthesis using bacterial and mammalian polymerases. The thought to be physiologically significant (2, 12). compound was inactive as a substrate for mammalian DXA In contrast to the results with E. coli DNA polymerase, York polymerase, bacterial DNA polymerase, mammalian RNA and LePage (18) recently reported that mammalian DNA poly polymerase, and bacterial RXA polymerase. The compound was merase (from TA 3 ascites cells) was noncompetitively inhibited found to inhibit DNA synthesis catalyzed by DNA polymerase by ara-ATP. This report prompted us to reexamine the effect of obtained from both calf thymus and bovine lymphosarcoma; the ara-ATP on the DNA-dependent polymerases. We have con results suggest a form of mixed inhibition between ara-ATP and firmed the finding of York and LePage that the mammalian deoxyadenosine triphosphate. No significant inhibition of Esch- enzyme is inhibited by ara-ATP; however, the inhibition appears erifhia coli DNA polymerase was observed, and RNA poly to be partially competitive with deoxy-ATP. We have also merases of both bacteria and mammalian cells were not inhibited shown that the E. coli enzyme is relatively insensitive to this by ara-ATP. In general, ribonucleoside triphosphates did not compound. inhibit DNA polymerase and deoxyribonucleoside triphosphates did not inhibit RNA polymerase. MATERIALS AND METHODS INTRODUCTION Preparation of ara-ATP. ara-A was synthesized by the D-Arabinosyl nucleosides inhibit the growth of both cultured method of Glaudemans and Fletcher (10). A portion was tritiated mammalian cells and bacteria, as well as the multiplication of by the Wilzbach procedure (17), by New England Nuclear Corp., DNA viruses in animal cells [see review by Cohen (4)]. While combined with 10 parts of nonradioactive ara-A and recrystal- the effect appeared to be primarily due to inhibition of DNA lized to constant specific activity. 5'-ara-AMP was prepared by synthesis in intact cells (3, 4, 15), earlier studies in this laboratory the Tener procedure (16). (The insolubility of ara-A required with nucleotides and enzymes of Escherichia coli catalyzing the addition of dimethyl formamide to assist in solubilizing the polynucleotide synthesis failed to elicit possible enzymatic sites nucleoside prior to the addition of the reagents.) The 5'-ara-AMP of inhibition. For example, Cardeilhac and Cohen (2) found that was separated on Dowex 1-C1 from unreacted ara-A, a cyclic bacterial DNA polymerase was not significantly inhibited by ara-AMP, and ara-A-polyphosphates. 5'-ara-AMP was eluted ara-CTP.3 Neither was RNA polymerase, and, although the in 33','r yield by 5 HIMHC1 followed by a cyclic ara-AMP in 11% yield. [Although the N to P ratio, Chromatographie prop erties, and ease of formation of the cyclic nucleotide suggested 1This investigation was supported by USPHS Grants 7005from that it might be 3',5'-cyclic ara-AMP, it was stable to heating the National Institute of Allergy and Infectious Disease and 10390 in Ha(OH)2 and may actually be the rt's-2',5'-cyclic ara-AMP from the National Institute of (Â¡eneralMedical Sciences. 2Research Career Development Awardee (GM-KS-12,888) of (see Ref. 4, p. 33)]. The 5'-ara AMP was isolated as the barium salt, converted to the USPIIS. 3The abbreviations used are: ara-A or arabinosyl adenine, the free acid with Dowex-50-H+, and dried in vacuo. The crystal 9-0-D-arabiiiofuraiiosyladeniiie; ara-AMP, ara-ADP, ara-ATP, line acid was dissolved in methanol and trioctylamine. The 9-0-D-aral)inofuranosyladenine-5'-mono-, di-, and -triphosphates; methanol was removed and dioxane and dimethylformamide ara-COP, ara-CTP, l-/3-D-arabinofuranosylcytosine-5'-di- and were used to dry the trioctylamine salt of ara-AMP. The latter triphosphates; UNA polymerase, nucleoside triphosphate: RNA was converted to the P'-ara-A-5'-P2-diphenylpyrophosphate by nucleotidyltransferase, EC 2.7.7.6; DNA polymerase, deoxy- the procedure of Michelson (13). The trioctylamine salt was dis nucleoside triphosphate: DNA deoxynucleotidyl transferase, EC 2.7.7.7. ; TTP, deoxythymidine-5'-triphosphate; deoxy-ATP, solved in 0.4 ml dimethylformamide plus 1.5 ml dioxane, and to deoxyadenosine-5'-triphosphate; ADP, adenosine-5'-diphosphate; this solution 0.2 ml dry tributylamine and 0.12 ml dry redistilled CDP, cytidine-5'-diphosphate; deoxy-GMP, deoxyguanosine-5'- diphenylphosphoryl chloride were added. After three hours at phosphate. room temperature, the solvent was removed and the oil caused to Received September 22, 190Â«;acceptedMay 2, 1967. congeal by the addition of anhydrous ether at 0Â°C.The ether

1528 CANCER RESEARCH VOL. 27

ara-ATP 4.8>tM ara-ATP

9.6/iM ara-ATP

19.2/iM ara-ATP

24 32 40 48 56 -0.05 0.05 0.10 0.15 _[_ S

8 16 24 Â¡(/Â¿M) CHART 1.4-C. Inhibition of DNA polymerase of lymphosarcoma by ara-ATP. The reaction mixture (0.25 ml) contained: 50 mM tris- (hydroxymethyl)aminomethene buffer, pH 8.1; 2 mm MgCU ; 2 mM 2-mercaptoethanol; 80 Â¿IMeachof dTTP, dCTP, and dGTP-"C (1.05 X IO6 cpm//umole) ; heated salmon DNA, 28.5 mamÃ³les of deoxynucleotide; varying concentrations of dATP and ara-ATP; and 200 fig of enzyme. After incubation for 60 min at 37Â°C,the reaction was terminated by the addition of 0.2 ml of 7% perchloric acid, albumin (1 mg) was added, and the acidified mixture centrifuged. The precipitate was washed twice with 3-ml portions of 1% perchloric acid, dissolved in 1.5 ml of 0.2 N NH4OH, decanted into metal planchÃ©is,dried, and the radioactivity measured in a Geiger-MÃ¼ller counter with Micro- mil window. No correction for self-absorption (approximately 25% in all samples) has been made. In Graphs 1/i and 1C, incorporation in the absence of dATP (0.06 mamÃ³le) was subtracted. Similar plots were obtained when incorporation in the absence of I)NA (<0.01 m/imole) was used as the "blank." Charts 1.4 and IÃŸ:â€¢ â€¢,no ara-ATP; O O, 1.9 AIMara-ATP; D D, 4.8 AIMara-ATP; V V, 9.6 Â¿Â«Mara-ATP;A A, 19.2 Â¿IMara-ATP. Chart 1C: â€¢ Â».11.2MMdATP; V V, 16.8AIMdATP; O O, 28.0ACMdATP. ara-ATP, 9-/3-D-arabinofuranosyladenine; dATP, dCTP, dGTP, and dTTP, 5'-triphosphates, respectively, of deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine, dGMP, deoxyguanosine-5'-phosphate.

SEPTEMBER 1967 1029

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. J. J. Furth and Seymour S. Cohen was decanted and the precipitate rewashed with ether. The TABLE 1 precipitate was redried by the addition and evaporation of Effect of ATP" on DNA Polymerase of Lymphosarconia dioxane and a solution of tributylamine pyrophosphate (750 The reaction mixture was as described in the legend to Chart 1. AmÃ³lesin 1 ml) and 0.6 ml dry pyridine were added. The mix The concentration of dATP was 16.8 AIM;the concentration of ATP ture was shaken gently at room temperature for 45 minutes and is specified in the table. After incubation for 30 minutes at 37Â°C, the pyridine removed in vacuo. The reaction mixture was dis incorporation of radioactive substrate into acid-insoluble form solved in water and applied to a Dowex 1-C1 column. ara-ADP was determined as described in the legend to Chart 1. was eluted, in 6% yield, with a solution containing 10 HIMHC1 (â€žM)ATP09.Concentration and 25 HIMLiCl, and ara-ATP in 25% yield with a solution con acidincorporated(â€žâ€žmoles)168173188192 taining 10 HIMHC1 and 10 mia LiCl. The solution of ara-ATP was neutralized with tributylamine, taken almost to dryness, precipitated with ethanol and acetone (in which LiCl is soluble), washed with acetone, and dried. The G38.476.8dATP16.816.816.816.8Deoxyguanylic product contained 2.95 Â¿miÃ³lesorganicP per /wnole adenine, assuming ara-ATP to have the extinction coefficient of ATP. The ratio of acid-labile P to total P was 0.69 and the product " ATP, adenosine-5'-triphosphate ; dATP, deoxyadenosine-5'- contained less than 0.05 /umole Pi per mole ara-ATP. The com triphosphate. pound was hydrolyzed completely to ara-A by rattlesnake venom and is therefore considered to be entirely the 5'-phosphate. Preparation of Mammalian Polymerases. Tissue was disrupted in a Waring Blendor and the crude extract centrifuged at 100,000 X g. Protamine sulfate was added to the supernatant solution and the precipitate collected by centrifugation. DNA polymerase was extracted from this pellet with 0.1 M sodium Buccinate, pH 6, and concentrated by precipitation with (NIL.)2- SÃœ4.RNA polymerase was then extracted from the pellet with 0.5 Msodium succinate, pH 6. The preparation and properties of RXA polymerase have been described previously (6). The preparation of DNA polymerase used in these experi ments, unless otherwise specified, was obtained from bovine lymphosarcoma. Incorporation of radioactive precursor into acid-insoluble material was completely dependent on the addition of denatured DNA and metal ion, and dependent on a full comple ment of deoxytriphosphates for optimal activity. If one triphosphate was omitted incorporation of labeled substrate varied from 10 to 15% of the incorporation observed in the complete system. The rate of reaction was proportional to time for approximately 120 minutes and the product was sensitive to DNase (9). E. coli RNA polymerase was obtained as described previously (8). E. coli DNA polymerase, obtained as a by-product of this proce dure, was further purified by chromatography on diethylamino- ethyl cellulose (11). RNA polymerase was assayed as described 01234 previously (6). dATP(fiM) The assay of DNA polymerase is described in the legends to CHART 2. Effect of ara-ATP and ATP on E. coli DNA poly- Charts 1 and 2. Labeled nucleoside triphosphates-14C and -3H merase. The reaction mixture (0.5 ml) contained: 50 HIMtris(hy- (other than ara-ATP-3H) were obtained from Schwarz BioRe- droxymethyl)aminomethane buffer, pH 8.1; 4 HIM MgClj ; 20 search. Calf thymus and salmon DNA were obtained from AIMeach of dCTP and dTTP; 10 MMdGTP-14C (1.13 X 10Â«cpm/ Worthington Biochemical Corporation and Sigma Chemical gniole); heated calf thymus DNA, 40 m/mioles of deoxymicleotide; Company, respectively. DNA was denatured by heating at and varying concentrations of dATP. The concentrations of ara- 100Â°Cfor4 minutes followed by cooling in an ice bath. ATP and ATP, when added, were 24 JIM.The reaction was initiated by the addition of enzyme (128 Â¿ig)and terminated after 20 min at 37Â°C. RESULTS Incorporation of labeled substrate into acid-insoluble material The inhibition of lymphosarcoma DNA polymerase is shown was determined as described in the legend to Chart 1. V V, in Chart 1 A-C. As shown in Chart L4, increasing amounts of dATP only; A A, dATP + ATP; O O, dATP 4- ara- ATP. ara-ATP, O-iS-D-arabinofuranosyladenine-S'-triphosphate; ara-ATP resulted in reduced rate of incorporation of deoxy- ATP, adenosine-5'-triphosphate; dATP, dCTP, dGTP, and GMP into an acid-insoluble form. A linear time course has been dTTP, 5'-triphosphates, respectively, of deoxyadenosine, deoxy- intercepted at 60 minutes in the presence as well as absence of cytidine, deoxyguanosine, and deoxythymidine; dGMP, deoxy- ara-ATP. For example, in an experiment similar to that shown guanosine-5'-phosphate. in Chart 1, the incorporation with 14 pM deoxy-ATP was 75

1530 CANCKIÃŽliKSKAKCH VOL. 27

/temÃ³lesof labeled deoxy-GMP in 30 minutes and 138 /Â¿/Â¿moles TABLE 4 in 60 minutes. When 18.4 /Â¿Mara-ATPwas added, incorporation Effect of ara-ATP" and dATP on RNA Polymerase of Bovine in 30 and (50minutes was 38 and 73 /Â¿Â¿uÃ±Ã³les,respectively.Thus Lymphosarcoma it seemed feasible to take the data reported in Chart 1A and plot The reaction mixture (0.25 ml) contained: 50 HIMtris(hydroxy- the reciprocal of the velocity against the reciprocal of the sub methyl)aminomethane-maleate buffer, pH 8.1 ; 8 mM MgCl2 ; 2 HIM strate concentration at three levels of inhibitor. Straight lines MnCl2 ; 2 HIM 2-mercaptoethanol; 250 MMeach of ATP, CTP, could be drawn, intersecting between the ordinate and the ab- and GTP, and UTP-14C (3.00 X IO6 cpm/Mmole) ; dATP or ara- scissa (Chart IB). When the reciprocal of the velocity was plotted ATP as noted; native calf thymus DNA, IG.OmamÃ³les of deoxy nucleotide; and 450 Mg of enzyme. After incubation for 20 min as a function of inhibitor concentration at three levels of sub at 37Â°C,the reaction was terminated by the addition of 0.2 ml of strate, straight lines could also be drawn, intersecting between 7% perchloric acid and incorporation into acid-insoluble material determined as described in the legend to Chart 1. TABLE 2 Comparison of ara-A TPa and dTTP as substrates for DXA (mM)ATP0.250.250.250.250.250.250ara-ATP00.480.961.44000dATP00000.721.440UridylicConcentration acid incorpo Polymerase of Lymphosarcoma rated inmoles)0058575f,61617 The reaction mixtures (0.5 ml) contained: 50 mM phosphate buffer, pH 7.5; 2 mm MgClÂ«; 2 mM 2-mercaptoethanol; 160 /*M each of deoxycytidine-S'-diphosphate, deoxyguanosine-5'-triphosphate and either deoxyadenosine-5'-triphosphate plus dTTP-3H (915 cpm/m/zmole) or dTTP plus ara-ATP-3H (191 cpm/nifimole) ; heated calf thymus DNA, 44 mamÃ³les of deoxynucleotide; and 315 tig of enzyme. Incorporation of radioactive substrate into acid-insoluble form was determined as described in the legend to Chart 1 except that (a) no albumin was added, (6) acid-insoluble material was washed " ara-ATP, 9-$-D-arabinoftiranosyladenine-5'-triphosphate; three times with 1% perchloric acid, and (c) a windowless Geiger- dATP, deoxyadenosine-5'-triphosphate; ATP, CTP, GTP, and MÃ¼llercounter was used. No correction has been made for self- UTP, adenosine-, cytidine-, guanosine-, and uridine-, 5'-triphos- absorption. phates. Labeled deoxynucleotide incorporated (mamÃ³les) TABLE 5 min0 min1.04 Effect of ara-ATP" and dATP on RNA Polymerase of E. coli The reaction mixture (0.25 ml) contained: 50 mM tris (hydroxy- dTTP-ni (2 cpm) (956 cpm) methyl)aminomethane-maleate buffer, pii 8.1 ; 8 mM MgCU ; 2 mM ara-ATP -'H0 0 (4 cpm)90 <0.01 (14 cpm) MnCl2 ; 2 mM 2-mercaptoethanol ; 320 //M each of GTP and CTP; 125 Â¿IMUTP-14C (6.82 X IO6 cpm/pmole); ATP as noted; native " ara-ATP, 9-/3-D-arabinofuranosyladenine-5'-triphosphate; dTTP, deoxythymidine-S'-triphosphate. calf thymus DNA, 16 mamÃ³les of deoxynucleotide; and 42 Â¿igof enzyme. The concentrations of ara-ATP and dATP are noted in the table. TABLE 3 The reaction was terminated by the addition of 0.2 ml of 7% Inhibition of DXA Polymerase of Calf Thymus by ara-A TP" perchloric acid and incorporation into acid-insoluble material The reaction mixtures (0.25 ml) contained: 50 niM Tris buffer, determined as described in the legend to Chart 1. pH 8.1; 2 HIM MgCU ; 2 HIM 2-mercaptoethanol; 80 JUMeach of deoxythymidine-5'-triphosphate and deoxycytidine-5'-triphos- Uridylic acid incorporated (AmÃ³les) phate; 40 MM deoxyguanosine-5'-triphosphate-14C (1.13 X 10' ATP concentration(Mu)05.611.216.822.431.062.0Control112337435197139+ ara-ATP cpm/Vimole) ; heated salmon DNA, 13 mamÃ³les of deoxynucleotide ; (96Â»v,1228414062-(-dATP(96MM)1432314550 and 50 Mg <>fcalf thymus enzyme. The concentration of ara-ATP and dATP are noted in the table. The reaction was terminated by the addition of 0.2 ml of 7% perchloric acid and incorporation into acid-insoluble material determined as described in the legend to Chart 1.

(///Â¿moles)Control55224279410382395+acid incorporated dATP concentration(UM)05.210.412.224.448.8Deoxyguanylic ara-ATP (47/iu)325652103 0 ara-ATP, 8-/3-D-arabinof uranosyladenine-5'-triphosphate ; dATP, deoxyadenosine-5'-triphosphate; ATP, CTP, GTP, and UTP, 5'-triphosphates of adenosine, cytidine, guanosine, and uridine, respectively.

the ordinate and the abscissa (Chart 1C). These results suggest a form of mixed inhibition between ara-ATP and deoxy-ATP. In " ara-ATP, 9-/3-D-arabinofuranosyladenine-5'-triphosphate; contrast, in a similar experiment, ATP did not inhibit the system dATP, deoxyadenosine-5'-triphosphate. with deoxy-ATP as substrate (Table 1).

SEPTEMBER 1967 1531

In an attempt to see if this inhibition was the result of the zyme appears also to reject the epimer of ribose (i.e., arabinosyl- incorporation of arabinosyl nucleotide into DNA parallel incu nucleotides) in incorporation reactions at least to the levels of bation vessels were set up, with ara-ATP-3H in one tube and sensitivity determined by the design of the experiment. The TTP-3H in the other (Table 2). Under conditions in which 1.04 D-arabinosyl configuration appears to be more nearly akin to invÃnolesfrom TTP-3H were incorporated into acid-insoluble the D-deoxyribose configuration than does D-ribose, however, form, less than 0.01 m/umole were incorporated with ara-ATP-3H since ara-ATP is a better inhibitor than ATP with the mam as substrate. From these results it can be concluded that if ara- malian DNA polymerase, while ara-ADP approaches the in ATP is utilized at all, it is utilized at less than one-hundredth hibitory activity of deoxy-ADP with polynucleotide phospho- the efficiency of TTP. DNA polymerase of neoplastic tissue is rylase. not unique in being inhibited by ara-ATP, as similar results Finally, the inhibition of mammalian DNA polymerase, but have been obtained with enzyme purified from calf thymus not that from E. coli, points out the advisability of testing po (Table 3). tential tumor inhibitors on mammalian enzymes. In contrast to these results with mammalian DNA polymerase, E. coli DNA polymerase was not significantly inhibited by ara- ACKNOWLEDGMENTS ATP (Chart 2). RNA polymerases of lymphosarconm and E. We are indebted to Mrs. Patricia Ho for excellent technical coli were not inhibited by ara-ATP and were not inhibited by assistance. deoxy-ATP (Tables 4, 5). REFERENCES DISCUSSION 1. Berg, P., Fancher, H., and Chamberlin, M. The Synthesis of Mixed Poly nucleotides Containing Ribo- and Deoxyriho- These results confirm the inhibition of DNA polymerase by nucleotides by Purified Preparations of DNA Polymerase from ara-ATP observed by York and LePage (18). However, while EscherÃ¬chiacoli. In: H. J. Vogel, V. Bryson, and J. O. Lampen (eds.), Informational Macromolecules, pp. 467-483. New inhibition of DNA polymerase could be the mechanism by which ara-ATP inhibits DNA synthesis in cultured mammalian cells York: Academic Press, Inc., 1963. 2. Cardeilhac, P. T., and Cohen, S. S. Some Metabolic Properties (and tumors) (18), evidence suggesting other possible sites of of Nucleotides of 1-0-D-Arabinofuranosylcytosine. Cancer action has also been obtained. ara-ADP and ara-ATP inhibit Res.,24: 1595-1603, 1964. the conversion of ribonucleoside diphosphates to deoxyribo- 3. Chu, M. Y., and Fischer, G. A. Comparative Studies of Leu- nucleoside diphosphates by the mammalian ribonucleotide re- kemic Cells Sensitive and Resistant to Cytosine Arabinoside. ductase (14, 18), and, although possibly of lesser significance, Biochem. Pharmacol., 14: 333-341, 1965. ara-ATP inhibits some mammalian amino-acid-activating sys 4. Cohen, S. S. Introduction to the Biochemistry of o-Arabinosyl tems (A. C. Griffin, jiersonal communication). The failure to Nucleosides. Progr. Nucleic Acid Res., 5: 1, 1966. observe significant inhibition of E. coli DNA polymerase by ara- 5. Doering, A., Keller, J., and Cohen, S. S. Some Effects of D- ATP (although ara-A inhibits DNA synthesis and is lethal to Arabinosyl Nucleosides on Polymer Synthesis in Mouse Fibro blasta. Cancer Res., 26: 2444-2450, 1966. certain strains) emphasizes the difficulty in ascribing inhibition 6. Furth, J. J., and Ho, P. The Enzymatic Synthesis of Ribo- of DNA synthesis solely to inhibition of DNA i>olymerase. (The nucleic Acid in Animal Tissue. I. The Deoxyribonucleic Acid effect of ara-ATP nucleotides on the ribonucleotide reductase of Directed Synthesis of Ribonucleic Acid as Catalyzed by an E. coli is not yet known.) Enzyme Obtained from Bovine Lymphosarcoma Tissue. J. It may also be noted that no evidence was obtained for the Biol. Chem., 21ft: 2602-2606, 1965. incorporation of ara-ATP into the polydeoxyribonucleotide 7. Furth, J. J., and Ho, P. L. Enzymatic Synthesis of Nucleic synthesized by mammalian DNA polymerase (Table 2) or the Acid in Animal Tissues. Federation Proc., 25: 645, 1966. incorporation of ara-CTP into the polydeoxynueleotide synthe 8. Furth, J. J., and Pizer, L. I. Deoxyribonucleic Acid-dependent sized by E. coli DNA polymerase (2). Since animal cells inhibited Ribonucleic Acid Synthesis in Escherichia coli Infected with with ara-A or ara-(\ and bacteria killed by ara-A, can synthesize Bacteriophage T2. J. Mol. Biol., 15:124-135,1966. DNA at essentially normal rates after removal of the ara-A or 9. Furth, J. J., Rosenberg, M., and Ho, P. L. Comparison of the ara-C (4, 5), the inhibition of DNA synthesis is reversible and Requirements for Ribonucleic Acid Synthesis with the Re quirements for Deoxyribonucleic Acid Synthesis in Animal probably is not the result of terminal addition of arabinosyl Tissues. J. Cell Physiol., 69: 209-217, 1967. nucleotide to the DNA chain. 10. Glaudemans, C. P. J., and Fletcher, H. C., Jr. Synthesis with The failure to observe inhibition of RNA polymerase, both Partially Benzvlated Sugars, III. A Simple Pathway to a mammalian and bacterial, is consistent with the failure of D- "cis-Nucleoside," 9-/3-D-Arabinofuranosyladenine (Spongo- arabinosyl compounds to inhibit RNA synthesis in vivo (5). adenosine). J. Org. Chem., 28: 3004-3006, 1963. These experiments also emphasize the importance of 2' position 11. Hurwitz, J., Furth, J. J., Anders, M., and Evans, A. The Role of the sugar in the specificity of enzymes catalyzing polynueleo- of Deoxyribonucleic Acid in Ribonucleic Acid Synthesis. II. tide synthesis. RNA | olymerase not only requires a 2'-hydroxyl The Influence of Deoxyribonucleic Acid on the Reaction. J. Biol. Chem., 237: 3752-3759, 1962. but it must be in the cis position. A nucleoside triphosphate 12. Lucas-Lenard, J., and Cohen, S. S. The Inhibitory Effect of lacking the hydroxyl group (deoxyribose) or having it in the Substrate Analogues on Polynucleotide Phosphorylase. Bio- irons configuration (D-arabinose) is essentially inert in the RNA chim. Biophys. Acta, 123: 471-477, 1966. polymerase reaction. DNA polymerase, on the other hand, re 13. Michelson, A. M. Synthesis of Nucleotide Anhydrides by An jects a nucleoside triphosphate with a 2'-hydroxyl group (ribose), iÃ³nExchange. Biochim. Biophys. Acta, 91: 1-13, 1964. unless Mg'H" is replaced by Mn++ (1). In in vitro tests, the en 14. Moore, E. C., and Cohen, S. S., Effects of Arabinonucleotides

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on Kibonucleotide Reduction by an Enzyme System from Rat 17. Wilzhaeh, K. E. Tritium Labeling by Exposure of Organic Tumor. J. Biol. Chem., 242: 2116-2118, 1967. Compounds to Tritium Gas. J. Am. Chem. Soc., 79: 1013, 15. Silagi, S. Metabolism of l-/3-i>-Arabinofurnosylcytosine in L 1957. Cells. Cancer Res., 25: 1446-1453, 1965. 18. York, J. L., and LePage, G. A. A Proposed Mechanism for 16. Tener, G. M. 2-Cyanoethyl Phosphate and Its Use in the the Action of 9-0-o-Arabinofuranosyladenine as an Inhibitor Synthesis of Phosphate Esters. J. Am. Chem. Soc., 83: 159- of the Growth of Some Ascites Cells. Can. J. Biochem. Phys- 168, 1961. iol., 44: 19-26, I960.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. Inhibition of Mammalian DNA Polymerase by the 5′ -Triphosphate of 9-β-d-Arabinofuranosyladenine

J. J. Furth and Seymour S. Cohen

Cancer Res 1967;27:1528-1533.

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