[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 (¡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, 190«;acceptedMay 2, 1967. congeal by the addition of anhydrous ether at 0°C.The ether 1528 CANCER RESEARCH VOL. 27 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1967 American Association for Cancer Research. Inhibition of Mammalian DXA Polymerase by ara-ATP 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 ¡(/¿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 ¿IMeachof dTTP, dCTP, and dGTP-"C (1.05 X IO6 cpm//umole) ; heated salmon DNA, 28.5 mamóles of deoxynucleotide; varying concentrations of dATP and ara-ATP; and 200 fig of enzyme. After incubation for 60 min at 37°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 planchéis,dried, and the radioactivity measured in a Geiger-Müller 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 mamóle) 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 Iß:• •,no ara-ATP; O O, 1.9 AIMara-ATP; D D, 4.8 AIMara-ATP; V V, 9.6 ¿«Mara-ATP;A A, 19.2 ¿IMara-ATP. Chart 1C: • ».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, deoxy- cytidine, 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. Amólesin 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 37°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 („M)ATP09.Concentration and 25 HIMLiCl, and ara-ATP in 25% yield with a solution con acidincorporated(„„moles)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 ¿miólesorganicP 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.