RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II

JOURNAL OF VIROLOGY, Jan. 1972, P. 130-142 Vol. 9, No. 1 Copyright © 1972 American Society for Microbiology Printed in U.S.A. RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II. Directing Influence of RNA in the Reaction JONATHAN P. LEIS AND JERARD HURWITZ Department ofDevelopmental Biology and Cancer, Division of Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 30 September 1971 The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg2+, and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qf; and synthetic homopolymers such as polyaden- ylate polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3' hydroxyl ends of primer strands. The product is an RNA-DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S.

Ribonucleic acid (RNA) tumor viruses contain MATERIALS AND METHODS an enzyme which catalyzes the synthesis of deoxy- AMV was a generous gift of J. Beard, Duke Uni- ribonucleic acid (DNA) from RNA (3, 26). The versity, and RLV was obtained from The Pfizer Co., products include RNA DNA hybrids, single- Inc. Maywood, N.J. stranded DNA, and double-stranded DNA. The Q3 RNA was a gift from G. Kuo of this institution; DNA product is of low molecular weight and is MS2 RNA wam from Miles Laboratories, Inc.; puri- composed of sequences complementary to the fied E. coli tRNAf"et was a gift from L. Schulman; f2 viral RNA (8, 9, 22, 24). In addition to RNA- RNA was a gift from U. Maitra; and Rous sarcoma primed activity, the polymerase also catalyzes a virus (RSV) RNA was a gift from P. Duesberg, Uni- DNA-dependent DNA synthesis (9, 25). versity of California, Berkeley. Mung bean nuclease I (15, 17) was generously pro- In the preceding paper (14), the mechanism vided by M. Laskowski, Sr. This enzyme was dis- of DNA priming of deoxynucleotide incorpora- solved in 0.02 M tris(hydroxymethyl)aminomethane tion was examined. These studies indicated that (Tris)-hydrochloride (pH 7.5) before use. both avian myeloblastosis virus (AMV) and All other materials were obtained as described Rauscher leukemia virus (RLV) polymerases previously (14). catalyzed repair-like reactions and that the po- Assay of RNA-primed reactions. Unless otherwise lymerases were unable to initiate de novo new indicated, reaction mixtures (0.05 ml) contained: 1 DNA chains. These polymerases resembled DNA ,mole of Tris-hydrochloride (pH 8.0), 0.5 ,tmole of polymerase II isolated from Escherichia coli in MgCl2, 0.25 ,umole of KCI, 0.3 ,umole of dithioerythreitol, 5 nmoles each of deoxyadenosine triphos- their priming requirements as well as their prod- phate (dATP), deoxyguanosine triphosphate (dGTP), ucts. and deoxycytidine triphosphate (dCTP), 1 nmole of In the present communication, the viral polym- 3H-deoxythymidine triphosphate (dTTP; 50 to 8,000 erases have been studied by using RNA primers. counts per min per pmole), and RNA and polymerase Evidence will be presented that the viral polym- as indicated. The reaction was stopped after 30 min of erases are unable to initiate DNA chains with incubation at 38 C by the addition of 0.1 ml of 0.1 M in RNA- sodium pyrophosphate, 0.02 ml of denatured salmon RNA primers. The products formed sperm DNA (2.6 ,umoles/ml), and 5% tricholoro- primed systems are RNA -DNA hybrids in which acetic acid. After 5 min at 4 C, the precipitate was polydeoxynucleotide chains are covalently at- collected on Gelman type E glass fiber filters, dried, tached to the RNA. and counted in 10 ml of toluene scintillation fluid in a 130 VOL. 9, 1972 RNA-DEPENDENT DNA POLYMERASE. II 131

scintillation counter. The following procedure was gradient in buffer A. The RNA in the 60S region of used to reduce the blank values when products were the gradient was pooled and then precipitated with collected from mixtures with deoxynucleoside tri- two volumes of 95% ethanol. The precipitate was phosphates of high specific activity (8,000 to 10,000 collected by centrifugation at 105,000 X g in a Spinco counts per min per pmole). Reaction mixtures as SW65 rotor for 60 min, and the RNA was dissolved described above were treated with 0.1 ml of 0.1 M in 20 mm NaCl, 0.1 mm EDTA, 20 mm sodium phos- sodium pyrophosphate, 0.05 ml of denatured salmon phate buffer (pH 7.0) and stored frozen at -10 C in sperm DNA (2.6 Mmoles/ml), 0.8 mg of serum al- small portions. bumin, and 2 ml of 5% trichloroacetic acid. After 5 Preparation of 3H-RLV RNA. Tritium-labeled RNA min at 4 C the precipitate was pelleted by centrifuga- was prepared from RLV isolated from incubation of tion at 10,000 X g for 2 min in an International chronically infected mouse CLI cells adapted to refrigerated centrifuge, and the supernatant was dis- suspension culture (using an unpublished method carded. The precipitate was dissolved in 0.5 ml of 1 M developed by R. Bases of this institution) with 3H-uri- NH40H, reprecipitated with 5% trichloroacetic acid, dine (2 Ci/mmole; 2.5 nmoles/ml) for 18 hr. Virus was collected, and counted on glass fiber filters as de- purified by isopycnic banding in sucrose, and RNA scribed above. was extracted and prepared as above. These prepara- Assay of ribonuclease activity. Ribonuclease was tions were carried out in collaboration with R. Bases. assayed in a volume of 0.05 ml containing: 2.5 Mmoles Equilibrium density gradient centrifugation in of Tris-hydrochloride (pH 8.0), 0.5 ,umole of MgC12, Cs2SO4 solution of DNA polymerase product treated 0.25 ,umole of dithioerythreitol, 0.25 jAmole of KCI, with or without formaldehyde. A 3H-labeled product 0.12 A260 3H-poly uridylic acid (U) (7 X 104 counts/ containing 7.1 X 104 counts/min in 0.5 ml was min), and enzyme. After incubation at 38 C for 30 treated with 0.5 ml of phenol neutralized to pH 8.0; min, 0.125 ,mole of denatured salmon sperm DNA, the aqueous phase was collected and again shaken 0.8 mg of albumin, and 0.5 ml of 5% trichloroacetic with phenol; the combined phase was dialyzed for 3 acid were added; the tubes were placed in ice for 5 min hr against 0.1X SSC (SSC = 0.15 M NaCl + 0.015 M and then centrifuged for 2 min at 10,000 X g in an In- sodium citrate). Samples (0.1 ml) were added to 2 ml ternational refrigerated centrifuge. The supernatant of 0.02 M sodium phosphate buffer (pH 7.0) contain- fluid containing acid-soluble materials was counted in ing or lacking 0.25 M HCHO. The solutions containing 10 ml of Bray's (4) scintillation fluid. formaldehyde were heated at 85 C for 10 min and Gradient centrifugation assay for ribonuclease ac- then cooled quickly. Solid Cs2SO4 (2.6 g) was added; tivity. To assay for ribonuclease, 1.4 nmoles of 3H-f2 the solution was diluted to 4 ml with 4X SSC and RNA (6,000 counts/min) was incubated under the centrifuged in polyallomer tubes for 44 hr at 38,000 conditions for the DNA polymerase assay with the rev/min in an SW39 rotor. Fractions were collected labeled deoxynucleoside triphosphate omitted. After from a hole pierced in the bottom of the tube, re- incubation, the reaction mixture was brought to a fractive indexes were measured, and the amount of final concentration of 0.1 M ethylenediaminetetra- acid-insoluble radioactivity was determined. 3H-poly acetic acid (EDTA), 0.4% sodium dodecyl sulfate (U) and 3H-d(AT) copolymer markers were run in (SDS), and 3% formaldehyde in a volume of 0.25 ml; different tubes. heated to 65 C for 15 min; and layered on a 5-ml 5 to Equilibrium density gradient centrifugation of 20% linear sucrose gradient containing 0.2% SDS, poly (A) poly (U) polymerase product. Reaction mix- 3% formaldehyde, 20 mm sodium phosphate (pH 7.0), ture (0.1 ml) contained 3.7 nmoles of ct32P-dTTP 1 mM EDTA, and 0.1 M NaCl. The gradient was cen- (8,400 counts per min per pmole), 10 ,ug of albumin, trifuged at 300,000 X g for 240 min at 20 C in a 5 Mmoles of Tris-hydrochloride buffer (pH 8.0), 0.2 Spinco SW65 rotor. Thirty-drop fractions were col- jAmole of MnCl2, 20 nmoles of poly (A), 0.6 nmole of lected directly into counting vials containing 10 ml of poly (U) (average chain length 70), and 0.08 unit of Bray's scintillation fluid. When 3H-RLV RNA (0.15 RLV polymerase (phosphocellulose eluate). After nmole; 2,000 counts/min) was used instead of 3H-f2 30 min at 38 C, reactions were stopped by the addition RNA, the procedure was the same except that gradi- of 0.9 ml of SSC and 1 ml of neutralized aqueous ents were centrifuged at 180,000 X g for 2 hr in a phenol. The aqueous phase was dialyzed for 12 hr Spinco SW65 rotor. against 250 ml of SSC yielding 2.6 X 106 counts/min Preparation of AMV RNA. AMV (250 mg) was of acid-insoluble 32p. A portion was heated with pelleted in a Spinco 40 rotor at 105,000 X g for 30 HCHO as follows: 0.05 ml of 32p product was added min. The virus was suspended in 10 ml of 0.1 M NaCl, to a solution (2.95 ml) containing 10,moles of sodium 40 mm sodium phosphate (pH 7.0), and 0.1 mm phosphate buffer (pH 7.0), 500,umoles of HCHO, and EDTA (buffer A) and extracted four times by the 1 nmole of 3H-poly (U). The mixture was heated at phenol-SDS method described by Duesberg (5) except 75 C for 10 min and then quickly cooled in an ice- that bulk E. coli tRNA (1 optical density unit per ml) water bath. To the was added as a carrier. The RNA was precipitated by formaldehyde-treated preparation, the addition of two volumes of 95% ethanol, sus- 0.6 ml of lOX SSC and 2.6 g of solid Cs2SO4 were pended in 1 ml of buffer A, and precipitated with two added, and the mixture was centrifuged in polyallomer volumes of 95% ethanol. The precipitate was dried tubes at 36,000 rev/min in an SW39 rotor for 60 hr in vacuo and dissolved in 0.2 ml of buffer A, and the at 20 C. Fractions were collected after a hole was RNA was sedimented at 180,000 X g in a Spinco pierced in the bottom of the tube, refractive indexes SW65 rotor for 60 min at 4 C on a 5 to 20% sucrose were measured, and the amount of acid-insoluble 132 LEIS AND HURWITZ J. VIROL. radioactivity was determined. The recovery of 32p in approximately 90 to 95&X0 of ribonuclease activities the fractions counted was 72%7(. present in crude extracts were removed after passage Poly (A) -poly (U) transfer experiments. Reaction through the phosphocellulose column (pH 8.6). The mixture (0.1 ml) contained 0.8 nmole of a32P-dTTP remaining ribonuclease activity, measured with 3H- (1,700 counts per min per pmole), 10 ,ug of albumin, poly (U), was separated from the polymerase by sedi- 10 ,umoles of Tris-hydrochloride (pH 8.0), 0.1 ,mole mentation in glycerol gradients. The AMV polymer- of MnCI2, 28 nmoles of poly (A) and 2.9 nmoles of ase was estimated to have a molecular weight of poly (U) (average chain length 50 nucleotides), approximately 160,000 and sedimented more rapidly 5 ,umoles of 2-mercaptoethanol, and 1.67 Kornberg than ribonuclease activity. The enzyme activity ob- units of DNA polymerase I or 2.1 units of Rauscher tained after glycerol gradient centrifugation will be polymerase, or 0.38 unit of AMV polymerase. Reac- referred to as purified polymerase pl. Although puri- tions were incubated for 60 min at 30 C, carrier DNA fied polymerase pl was found to be free of ribonuclease (0.02 ml of salmon sperm; 2.6 Amoles/ml) was added, when this activity was measured employing 3H- and the precipitate was collected by centrifugation. poly(U), ribonuclease activity was detected when the The pellet was dissolved in 0.2 ml of 0.3 N NH40H, polymerase preparations (0.4 unit) were incubated acidified with trichloroacetic acid, and centrifuged. with 3H-RLV or 3H-f2 RNA preparations and the This procedure was repeated two more times, and the RNA species were subjected to formaldehyde dena- final pellet was dissolved in 0.5 N KOH. The amount turation. Under these conditions, a decrease in the of acid-insoluble radioactivity observed was 9.54 X sedimentation value of the labeled RNA was detected 105, 2.54 X 105, and 3.5 X 104 counts/min for reac- in sucrose gradients. Presumably, these RNA prepa- tion mixtures containing DNA polymerase I, RLV rations contain enough secondary structure so that polymerase, and AMV polymerase, respectively. The nicks introduced by ribonuclease do not change their solution was incubated for 22 hr at 37 C, after which sedimentation properties unless their secondary time 0.02 ml of salmon sperm carrier DNA was structure is destroyed. added. This was followed by acidification with HCl. AMV polymerase preparations containing no The pellet was collected by centrifugation, and the detectable ribonuclease activity were prepared by supernatant fluid was adsorbed to Norite (0.1 ml of chromatography of the phosphocellulose pH 8.6 10%). The Norite was washed twice with water, and eluate on a second phosphocellulose column at pH the adsorbed material was eluted three times with 7.0. The elution of the enzyme activity was carried 0.5-ml portions of 50% ethanol in 1.5 N NH40H. The out as previously described (14) with the exception ethanolic-NH3 eluates were concentrated to dryness that 0.04 M sodium phosphate buffer (pH 7.0) was and dissolved in water. The amount of 32P-labeled used in place of Tris buffer. Such polymerase prepa- nucleotide recovered at this point was: 10,720, 1,560, rations obtained in 60% yield will be referred to as and 720 counts/min from reactions containing DNA purified polymerase pll. Incubation of 0.25 unit of polymerase I, RLV polymerase, and AMV polym- AMV polymerase pll with 3H-f2 RNA did not alter erase, respectively. Samples (4,960, 1,250, and 520 the sedimentation profile in formaldehyde-sucrose counts/min from the DNA polymerase I, RLV, and gradients. AMV polymerase mixtures, respectively) with carrier 2'(3') mononucleotides were subjected to electrophoresis on Whatman 3 MM paper saturated with RESULTS 0.05 M sodium citrate, pH 3.5, for 1.5 hr at 6,000 v. The areas corresponding to the ultraviolet-absorbing Influence of enzyme concentration on rate of regions were cut out, eluted, and counted in Bray's DNA synthesis. The influence of enzyme concen- scintillation fluid. No 32p was detected in regions of tration on RNA-primed DNA synthesis was the electropherogram between the ultraviolet-ab- studied by using AMV RNA as primer (Fig. 1). sorbing regions, nor was there any 32p located at the As shown, a sigmoidal curve was obtained which origin. A small amount of 32p possibly representing reached a plateau at 20 ng of protein. This sig- di- or trinucleotides, etc. was detected ahead of the moidal relationship was also observed with DNA 2'(3') uridine monophosphate (UMP) region. primers. These results suggest that the AMV The radioactivity eluted from the 2'(3') UMP be of subunits region was subjected to the action of alkaline phos- polymerase may composed which phatase or crude snake venom (Crotalus adamanteus). tend to dissociate at low protein concentration In the former case, all of the 32p was rendered Norite (18), though other interpretations are possible. nonadsorbable, whereas in the latter case 90%/0 of the The amount of enzyme used in these experiments 32p remained Norite adsorbable. These observations was capable of incorporating 62 pmoles of de- indicate that the 32p in the 2'(3') UMP region is not oxynucleotides with poly d(AT) as primer under present in TpT or oligonucleotides. the standard assay conditions described in the Viral polymerase preparations. The DNA polym- preceding paper (14). The low activity observed erase activities of AMV and RLV were purified ex- with AMV RNA as primer is due to its limiting tensively from crude detergent extracts, utilizing poly concentration in the reaction. Reactions con- d(AT) copolymer as primer (14). Enzyme preparations of specific activity greater than 3 Amoles/mg of taining 0, 200, 400, and 1,000 pmoles of AMV protein were employed. In most of the experiments RNA resulted in the incorporation of <0.2, described below, the AMV polymerase was used. 5.9, 9.4, and 31.4 pmoles of deoxynucleotides, With this enzyme preparation, deoxyribonuclease and respectively. In all of the experiments presented VOL. 9, 1972 RNA-DEPENDENT DNA POLYMERASE. II 133

TABLE 1. Requiremeiits for synzthesis of DNA with 0 0 E avian myeloblastosis virus (AMV) polymerasea z Deoxynucleotide 0 Additions incorporation IX- (pmoles/30 min) 6 Complete...... 1.13 0 o Omit MgCl2 ...... <0.02 z 4 Complete + MnCl2 (1 mM) ...... 1. 6 w Complete - MgC12 + MnCl2 (I mM) ...... 0.63 0 2 / -j Complete - KCl ...... 0.96 O Complete - dithioerythreitol 0.20 z 0Qy Complete - RNA...... <0.02 0 0 2 4 6 Complete + ribonuclease (2,ug). <0.02 0 Complete + deoxyribonuclease PROTEIN (pg x 102) (10 jg) ...... 0.05 FIG. 1. AMV RNA-depetndenit DNA polymerase activity. AMV RNA (206 pmoles) was incubated with a Conditions of the polymerase assay were as 3H-diTP (8,000 counts per mini per pmole) under the described in the text with 67 pmoles of AMV RNA, conditions described in the text with varying concen- 0.2 unit of purified polymerase pll, 3H-deoxycyti- trations of purified AMV polymerase p1 as indicated. dine triphosphate (4,460 counts per min per pmole) and unlabeled deoxyadenosine triphos- below, limiting amounts of viral RNA were used phate, deoxyguanosine triphosphate, ance deoxy- because of our limited supply. thymidine triphosphate. Duesberg et al. (7) have shown that during purification of the polymerase from RSV there than was observed with either cation alone. The is no separation of DNA- and RNA-primed polymerase required the presence of sulfhydryl activities. We have obtained similar results with reagents such as dithioerythreitol or mercapto- AMV and RLV polymerases. Heat inactivation ethanol for activity and possessed a broad pH studies with the AMV polymerase indicate that optimum between pH 7.8 and 9.0, with maximum polymerase activity with either DNA or RNA activity observed at pH 8.2; deoxynucleotide primers is inactivated at the same rate. For incorporation at pH values of 9.8 and 7.2 was example, when AMV polymerase preparations 50% of that observed at pH 8.2. When RNA was were incubated for 3 min at 28, 44, 60, and 65 C, omitted from the incubation mixture, no de- nucleotide incorporation measured with AMV tectable deoxynucleotide incorporation occurred. RNA (206 pmoles) as primer was found to be Similarly, the addition of either pancreatic ribo- 5.6, 4.3, 2.0, and <0.2 pmoles in 30 min, respec- nuclease or deoxyribonuclease inhibited DNA tively; deoxynucleotide incorporation with d(AT) synthesis (Table 1). In another series of experi- copolymer (4.9 nmoles) as primer was found to ments, it was shown that AMV RNA-primed be 104, 80, 32, and 0.9, respectively, for these synthesis depended on the presence of all four preparations. In each case, the rate of inactiva- deoxynucleoside triphosphates for maximal action of polymerase activity with AMV RNA or tivity (Table 2); the omission of a single deoxy- d(AT) copolymer as template was the same. nucleotide markedly reduced DNA synthesis. These results support the idea that one enzyme is Furthermore, all four deoxynucleotides were in- responsible for both activities. corporated in approximately equimolar amounts Requirements of RNA-primed reactions. The (Table 3). requirements for the synthesis of DNA with Specificity of DNA polymerase for RNA tem- AMV RNA as template are summarized in Table plates. A number of RNA preparations supported 1. The purified polymerase pIl required magne- deoxynucleotide synthesis with purified polym- sium ions for activity; optimal activity occurred erase preparations in agreement with results at 10 mM Mg1+, and the requirement for Mg2+ obtained by Duesberg et al. (unpublished data). could be partially met by Mn2+ (1 mM). In polym- Viral RNA isolated from RSV and RLV stimu- erase preparations contaminated with ribonu- lated deoxythymidine monophosphate (dTMP) clease, such as polymerase pl, the activity with incorporation to about the same extent as AMV Mn2+ was greater than with Mge alone; 0.2 mM RNA (Table 4). Mn2+ gave optimum activity. However, with Several RNA preparations from sources other either enzyme preparation when magnesium and than RNA tumor viruses were tested. Of these, manganese ions were both present, there was a f2, MS2, Q3, and bulk E. coli transfer RNA greater rate of deoxynucleotide incorporation (tRNA) preparations were active templates for 134 LEIS AND HURWITZ J. VIROL.

TABLE 2. Effect of omissioni of cleoxyiiiilceoside TABLE 4. Comparisont of template activities of triphlosphates oln DNA synithesis" different RNA preparations"s Deoxynucleotide Additions incorporation Deoxynucleotide (pmoles/30 min) Amt of incorporation RNA (pmoles/30 min) F;xjt RNA added" added Complete 1.60 (pmoles) Omit dATP, dGTP, dCTP 0.18 '\lg2+Mg~+)Mg2+NIn2+ Omit dATP 0.24 I~~~~~ Omit dGTP 0.39 I AMV 206 ! 3.6 9.6 Omit dCTP 0.28 RSV 500 5.2 11.2 RLV 150 1.1 6.9 ,, Conditions for assay were as described in the .f2 10,000 0.9 43.5 text except that 0.2 nmole each of deoxyadenosine 1,650 0.2 8.5 triphosphate (dATP), deoxyguanosine triphos- Q3 Bulk Escheric/hia 8,700 i 1.5 5.6 phate (dGTP), and deoxycytidine triphosphate coli tRNA (dCTP), 0.03 nmole of 3H-dTTP (8,200 counts per E. coli tRNAf 7,800 0.14 min per pmole), 206 pmoles of avian myeloblastosis virus RNA, and 0.35 unit of purified poly- II AMV 206 2.6 8.3 merase pl were added. MS2 242 0.08 0.77 Q3 237 0.28 1.05 TABLE 3. Ilicorporaltiol oflabeled leoxvnucleotides f2 150 0.22 1.73 hy avianI myeloblastosis viruts (AMV) polymerase" bulk E. coli 260 0.45 0.33 tRNA Deoxynucleotide MNfet Labeled deoxynucleotide added incorporation E. co/itRNAf 390 0.08 (p)moles/30 min) /I Purified avian myeloblastosis virus (AMV) 'H-dTTP 0.31 polymerase pl (0.35 unit) was incubated with dif- 'H-dGTP 0.28 ferent RNA preparations and 3H-deoxythymidine 'H-dCTP ...... 0.39 triphosphate (8,200 counts per min per pmole) in 'H-dATP 0.29 the standard DNA polymerase assay. Mn2+ (0.2 mM) was added where indicated. In the experi- *l Four separate reaction mixtures were prepared ments reported above, all DNA synthesis obtained with 67 pmoles of AMV RNA and 0.2 unit of puri- with any of the RNA primers was quantitatively fied polymerase pil in which each one contained inhibited by the addition of ribonuclease (2 j.g). one of the deoxynucleoside triphosphates labeled I Abbreviations: RSV, Rous sarcoma virus; with tritium. In these reactions 1 nmole of :H- RLV, Rauscher leukemia virus; tRNA, transfer deoxynucleoside triphosphate was present with 5 ribonucleic acid. nmoles each of the unlabeled deoxynucleoside triphosphates. The specific activities of the labeled deoxynucleoside triphosphates were: JH- plate activity. RSV and AMV RNA species were deoxythymidine triphosphate (dTTP; 8,210 counts tested after heat treatment and found to be active per min per pmole), 3H-deoxyguanosine triphos- (Table 5). Ten to 30eG of the template activity phate (dGTP; 1,600 counts per min per pmole), was retained when assayed in the presence of 1'H-deoxycytidine triphosphate (dCTP; 4,460 Mg2+, whereas 50 to 75 c7 of the template activity counts per min per pmole), and :H-deoxyadeno- was observed when assayed in the presence of sine triphosphate (dATP; 7,090 counts per min Mn + and Mg2+. per pmole). Extent of DNA synthesis. The extent of DNA synthesis with AMV RNA as primer was investi- the synthesis of DNA in the presence of Mn2+ and gated (Fig. 2). As shown, the yield of deoxynu- Mg2ft, whereas a purified preparation of E. coli cleotide incorporation was equivalent to 3,' of tRNAfmt was not. The efficiency of priming with the added RNA primer. The observed plateau these RNA preparations, however, was markedly was not due to inactivation of enzyme activity lower than that observed with tumor viral RNA during the incubation since the addition of more species. enzyme at 20 min did not further increase deoxy- The native conformation of the tumor viral nucleotide incorporation. The addition of more RNA preparations is disrupted by heating at 80 C RNA, on the other hand, resulted in an immedi- in low ionic strength buffers (2, 6, 10). This ate resumption of deoxynucleotide incorporation treatment results in an irreversible change in the which again reached the plateau corresponding sedimentation coefficient of the RNA species to 3% of the added RNA. These results show that from 70 to 30S and, as found by Duesberg et al. only a limited amount of deoxynucleotide can be (unpublished data), a marked decrease in tem- incorporated with a given amount of RNA. In VOL. 9, 1972 RNA-DEPENDENT DNA POLYMERASE. II 135

TABLI 5. Effect of heat deniaturationi oni priminig has been presented by Bader and Steck (2) who activ'ity of aviani myeloblastosis virus (AMV) antd observed that the 60 to 70S Rauscher RNA Rotis sarcoma virus (RSV) RNA preparationsa remains intact after action of endogenous viral small RNA Deoxynucleotide ribonuclease; upon denaturation, Amt of incorporation species (<20 to 305) were detected. RNA added RNA (pmoles/30 min) We investigated the structure of the Rauscher (pmoles) RNA by studying its susceptibility to attack by

Mg2+ + Mn2+ several nucleases. Tritium-labeled RLV RNA was rendered acid-soluble after incubation with AMV ...... 206 1 .13 1 .59 pancreatic ribonuclease (Table 6). This result AMV heated.206 0.40 0.80 suggests that the secondary structure of the RNA RSV...... 400 1 .32 1 .45 does not involve long stretches of complementary RSV heated.400 0.15 1.10 sequences such as those found with the duplex I______reovirus RNA. In addition, complex structures a WThere indicated, RNA were th dic preparations such as poly (A) poly (U) are also susceptible to heate d atat8080C forafore33min andaN d rapidlypra pidly cooled;tcool addi-add pancreatic ribonuclease attack under the condi- were as described in Table 1 with purified u e tions tins emplo (Table 6 polyr nerase pII (0.2 unit). tions employed (Table 6). The Neurospora and mung bean I nucleases (15, 17, 19) specifically degrade single-stranded 0 but not duplex structures of DNA. These en- E zymes also attack single-stranded regions of RNA z o_ in RNA duplexes or single-stranded regions of .9 -0 4RNA and DNA in RNA DNA hybrids. As /1°0 shown in Table 6, 3H-poly (U) is completely sus- 3 ceptible to the action of Neurospora and mung

z . bean nucleases, after annealing to poly (A), 3H- A 9I2 /*./l _ poly (U) was completely resistant to the nu- C A ^ cleases. In other experiments, RNA- DNA hy- z brids were formed with fd DNA and RNA po- 0 lymerase and isolated by gradient density banding

lD306Croo / 140 170170 2ob0200 in Cs2SO4. The RNA in such structures is resistant to Neurospora heat denaturation of TIMETIME (min) nuclease; (m4) the hybrids renders the RNA susceptible to this FIG 2. Kintetics of deoxyntucleotide inicorporatioln nuclease preparation. with A4MV RNA anzd DNA polymerase. Assay conidi- When 3H-RLV RNA was treated with either tiolis were as described int the text with 3H-dCTP bean Neurospora nucleases, approxi- (4,460 counlts per mii per pmole), 67 pmoles of AMV mately 50%O of the RNA remained acid-insoluble. aiid 0.375 utit of purified polymerase 11 added. * RNA, r After 20 miii of incubationi, anl additionial 67 pmoles of These results suggest that there is considerable AMV RNA or 0.15 unit polymerase pII was added. internal structure of the RNA, in accord with the Symb(ols: (0) deoxynucleotide inicorporationz; (0) de- previously reported observations (2, 6, 8, 10, 20, oxyiiuceleotide intcorporationt after additioii of RNA at 21, Duesberg et al., unpublished data). Upon heat 20 miu'1; (A) deoxynucleotide iiicorporatioii after addi- denaturation, 90%O of the RNA was susceptible tioii f enizyme at 20 miii. to Neurospora nuclease action, suggesting a marked disruption of the internal structure of other experiments (not shown) conducted under the RNA. The interpretation of the latter finding condiItions described in Fig. 2, the addition of is complicated by the presence of nicks in the riboniuclease (2 ,ug) at 20 min had no effect on 3H-RLV RNA. Approximately 50 to 60% of the the yiield of DNA synthesis. 3H-RLV RNA in our preparations sedimented Susiceptibility of Rauscher RNA to nuclease and slower than 20S after heat denaturation. Nicks its eflfect on template activity. The RNA from present in the RNA would yield structures in- RNA tumor viruses sediments in neutral sucrose capable of reversible denaturation if, for example, graditents with a sedimentation coefficient of 60 hair-pin structures existed in intact RNA. A to 70S (8, 20, 21). Denaturation by treatment diagrammatic representation is shown in Fig. 3. with (dimethyl sulfoxide, urea, or heat (2, 6, 10) The single-stranded regions of the AMV RNA reducies the sedimentation coefficient to 20 to 305 were shown to be essential for template activity and s:,maller materials, suggesting that the native by use of the Neurospora nuclease. The nuclease RNA is composed of pieces hydrogen-bonded was preincubated with AMV RNA for 10 min at togeti her. Further support for such a structure 38 C to degrade single-stranded regions. After 136 LEIS AND HURWITZ J. VIROL. heating at 65 C for 4 min to inactivate the nu- TABLE 7. Effect of Neurospora ntuclease treatment clease activity, deoxynucleoside triphosphates of avian myeloblastosis virus (AMV) RNAa and polymerase were added and the rate of DNA Deoxynucleotide synthesis was measured (Table 7). AMV RNA Treatment of AMV RNA incorporation (pmoles) TABLE 6. Susceptibility of Rauscher leukemia virus None ...... 1.13 (RLV) RNA and poly (U) to enzymatic digestiona Heated at 65 C ...... 0.80 Neurospora nuclease (0.02 unit) <0.10 Acid- RNA Treatment insoluble RNA (70) a AMV RNA (206 pmoles) was incubated with Neurospora nuclease at 38 C for 10 min. The reac- 3H-RLV RNA Ribonuclease (4 ,ug) <5 tion mixtures were heated to 65 C for 4 min to Neurospora nuclease 56 inactivate the nuclease and then incubated under (0.02 unit) the DNA polymerase assay conditions described Neurospora nuclease 43 in Table 1. (0.04 unit) Mung bean nuclease 51 which was preincubated without the nuclease I (0.2 unit) retained 70% of its activity compared to non-pre- Pancreatic deoxyribo- 93 incubated controls. On the other hand, RNA nuclease (10,g) preincubated with the nuclease did not prime RNA heated at 100 °C 13 DNA synthesis. Similar structural requirements for 3 min; Neuro- for single-stranded regions have been demon- spora nuclease (0.02 unit) added strated for DNA-primed DNA synthesis as pre- 'H-poly (U) Ribonuclease (4 jg) <2 viously reported (14). Neurospora nuclease <2 Characteristics of DNA polymerase product. The (0.04 unit) nature of product of the AMV DNA polymerase Mung bean nuclease I <2 and AMV RNA was examined by treatment with (0.2 unit) various chemical and enzymatic reagents. As Poly (A) + ribonu- 7 reported by others, the labeled product possessed clease (4 Ag) characteristics expected of DNA. It was resistant Poly (A) + Neuro- >98 to hydrolysis by heating at 100 C at pH 6.5 or in spora nuclease (0.04 unit) 1 M KOH but was completely converted to an Poly (A) + mung >98 acid-soluble form after heating in 1 N HCI. The bean nuclease 1 (0.2 product was resistant to digestion by ribonuclease unit) and quantitatively attacked by deoxyribonuclease (Table 8). After treatment with alkali, the labeled a Tritium-labeled RLV RNA (80 pmoles, 2,340 product banded at a density of 1.45 in Cs2SO4 counts per min) was incubated in reaction mix- solution characteristic of DNA. The alkali- tures (0.1 ml) containing 5 ,moles of tris(hydroxy- treated product had a sedimentation coefficient methyl)aminomethane-hydrochloride (pH 7.5), of 6 to 7S measured in alkaline sucrose gradients 0.5 1Amoles of MgCl2, and nucleases as indicated. 3H-poly (U) (7 nmoles, 62,000 counts per min) with single-stranded "4C-labeled fd DNA as a and poly (A) (10 nmoles) were added where indi- reference marker. This represents a molecular cated, and reaction conditions were the same as weight of approximately 1.7 X 105 to 2.6 X 105. those described with RLV RNA. In reaction mix- The size of the DNA was the same whether it was tures treated with mung bean nuclease I, the poly- synthesized in the presence of Mg2+ and Mn2+ or nucleotides as indicated were added to solutions Mg24 alone. containing MgC12. After mixing, potassium acetate Product as an RNA- DNA hybrid. Duesberg buffer (pH 5.1; 2,Amoles) and enzyme were added. et al. (7) and Faras et al. (12) have reported that All other conditions were as described above. newly synthesized DNA formed with purified polymerase and RSV RNA cosediments with 60 A. Intact hair-pin heating cooling to 70S viral RNA in neutral sucrose gradients. -C UiCL We repeated this experiment with 3H-RLV or AMV RNA preparations with similar results. A of the 32P-labeled DNA was found B. Nicked hair-pin henoturooion small portion , I;I-[-*rT-----rT- -- no renaturation at the top of the gradient presumably due to the FIG. 3. Diagrammatic representation of hairpin presence of ribonuclease in the polymerase pl structures in RNA. The region designated with a bar preparation used. These results suggest that the represents complementary sequences. immediate polymerase product is an RNA DNA VOL. 9, 1972 RNA-DEPENDENT DNA POLYMERASE. II 137

TABLE 8. Euzymatic digestiont studies otn cq- DNA polymerase producta E Amt of product remaining acid- insoluble (%) in w Treatment of product expt i- 0 .1.919EI'l I II III cr 0 F- C.)z Ribonuclease (4-20,ug) ...... 100 100 .1.8 zCo Deoxyribonuclease (4 ,ug)...... <5 <5 I 0"w Neurospora nuclease 0.01 unit... 74 I 0.02 unit... 67 78 to 0.04 unit... 54 52 57 Mung bean nuclease I 0.2 unit 88 5 10 15 20 25 1.0 unit 70 FRACTION NUMBER Heat at 100 C for 5 min followed by: FIG. 4. Equilibrium CsCl solution gradient centtrif- Neurospora nuclease (0.04 ugation of DNA polymerase product. A DNA product unit) ...... 22 21 formed with AMV RNA as described in Fig. 5 was used Mung bean nuclease I (0.2 for CsCI solution gradienzt cenitrifugationi. The sample unit).44 was diluted to 2.1 ml with 2X SSC, and 2.8 g of solid Mung bean nuclease I (1.0 CsCI was added, yielding a density of 1.799. Centrifuga- unit).33 tion anid conditions were as described in Fig. 5. Sym- Heat at 100 C for 7 min in 1 M bols: (0) tritium-labeled product; (a) density. KOH followed by: Neurospora nuclease (0.04 Covalent attachment of the RNA to DNA. It unit) ...... 5 10 was previously reported (14; Leis and Hurwitz, Mung bean nuclease I (1 unit). 8 Fed. Proc. 30:1153, 1971) that the Rauscher and AMV DNA polymerases were incapable of a Products were prepared with avian myeloblastosis virus RNA (206 pmoles) and polymerase initiating synthesis of new chains with poly d(AT) pl as described in Table 4. Products were dialyzed copolymer as template. Deoxynucleotide in- to remove deoxynucleotides and then incubated corporation occurred at the 3'-OH end of a primer in 0.1 ml containing 5 ,umoles of Tris (hydroxy- strand, and all newly synthesized product was methyl)aminomethane-hydrochloride (pH 7.5), convalently attached to the poly d(AT) copolymer 0.5 gmole of MgC92, and enzymes as indicated. primer. We therefore investigated the nature of After 30 min at 38 C, the amount of acid-insoluble the linkage between the AMV RNA and the DNA radioactive product was measured. In each of these in the hybrid product. If RNA is covalently experiments, approximately 1,500 counts per min attached to corresponding to 5.7 pmoles of DNA product the DNA, then the two components were used. In reaction mixtures containing alka- should be inseparable by density gradient centri- line hydrolyzed product, the KOH was removed fugation after denaturation in the presence of with Dowex 50 (H+) before addition of the Neuro- formaldehyde. A 3H-dTMP-labeled product was spora nuclease or mung bean nuclease I. In all prepared (see above) and divided into two equal experiments carried out with the latter enzyme, portions. One half was heated at 85 C for 10 min the conditions described in Table 6 were used. in 0.25 M formaldehyde and the other sample was unheated. Both products were then analyzed by hybrid. This has been shown to be correct by the Cs2SO4 solution equilibrium density gradient following observations: (i) the majority of the centrifugation with 3H-poly(U) and 14C-T7 DNA 3H-dTMP-labeled product was found to be re- as density markers. The untreated product sistant (55 to 80%) to digestion by Neurospora or banded as a broad peak at densities intermediate mung bean I nucleases (Table 8). After the to that of free DNA and RNA (Fig. SA). This product was treated with alkali to digest RNA, heterogeneity would be expected for RNA DNA between 5 and 10% of the DNA was resistant to hybrids possessing varying amounts of the two Neurospora or mung bean I nucleases; (ii) the components. The product heat-denatured in dTMP-labeled product banded at a density of formaldehyde also banded at a hybrid density 1.835 in CsCl solution which is characteristic of (Fig. SB); no detectable product banded at the RNA (Fig. 4), whereas less than 2% banded as density of free DNA, indicating that under the free DNA. These experiments indicate that the conditions employed all of the DNA product was product, under the conditions employed, is covalently linked to RNA. (Similar denaturation mostly, if not exclusively, an RNA- DNA hybrid. experiments have been carried out with 138 LEIS AND HURWITZ J. VIROL. synthetic polynucleotides poly (A) poly (U), as well as with phage RNA f2. 3P-dTMP-labeled polymerase products were prepared with 3H-f2 RNA in the presence of Mn2+ (see above). One- half of the product was heated at 65 C for 15 min 0N / \ao in 3 formaldehyde, and both products were 6 sedimented through a neutral SDS-sucrose ~ ~~ /1.8 gradient. Formaldehyde was included in the E 4 gradient containing the heated product. The 32P_ 9 ~ ~~ ~ ~ 0 ~~~~~~~~~~^ labeled product cosedimented with the 3H- labeled f2 RNA before and after the formalde-

08 1.6 hyde treatment (Fig. 6), indicating that the DNA 1.4 was covalently linked to the f2 RNA. Similar

O 5 10 15 20 experiments with 3H-RLV RNA as template were 4B performed. There was general correspondence U 8 Z o) between the sedimentation profile of 32P-DNA ~~~~~2.00a and 3H-RNA. A sizeable amount of the RNA however, did not cosediment with the 32P-DNA 6 a. after formaldehyde denaturation, most likely due I~~~~~~1.8 'lb to the presence of nicks in the 60 to 70S RNA. .~4 In the absence of formaldehyde treatment, 3H- ~~~~~~~~~~~~~~~~~~~1.6and 32P-labeled materials cosedimented as previously discussed.

1.4 The synthetic homopolymers, poly (A) -poly (U), in the presence of Mn2+ specifically support

0 5 10 15 20 the synthesis of poly(dT) as reported by Scolnick et al. (23). The product poly(dT) formed in such FRACTION NUMBER reaction mixtures banded in Cs2SO4 solution as a FIG. 5. Equilibriuim Cs2S04 solutiont grclienit celi- sharp peak at the density of 1.52 (Fig. 7B). trifiugcatio,l of DNA polymeraise produict. (A) A polym- 3H-poly(U) and 3H-poly(dT) added to separate eraise product (2.1 X 104 counts/mil) wvas prepalred gradients as standard markers banded at a with AMV RNA (206 pmoles) a,id 0.2 iuniit purified density of 1.66 (Fig. 7) and 1.43, respectively. polymerase p1, dialyzed against SSC, anid extracted After heat denaturation in formaldehyde, the with pheniol. A portioni was sedlimelited to equiilibrium in 32P-product still banded at 1.520 (see above) CS2SO4 solutionz for 44 hr at 123,000 X g as described in the text. The recovery of 'P was 71c (B) A portioni indicating covalent attachment of the poly(dT) to of the polymerase produict was heated in 0.25 Mi form- the RNA primer. The covalent attachment of aldehyde at 85 C for 10 mimi anid sedimenited to equiilib- 32P-poly(dT) to poly (U) was further demon- rium ill formaldehyde CS2S04 solultioIi as above. The strated by subjecting 32P-poly(dT) products to recovery of 32p was 66%". Symbols: (0) 3P-dTMP- alkaline hydrolysis. If 32P-dTMP is covalently labeled product; (0) denlsity. attached to poly (U), alkaline hydrolysis should result in the transfer of 32p to UMP residues RNA- DNA hybrids formed with E. coli DNA-de- yielding 32P-labeled 2'(3')UMP. A poly(dT) pendent RNA polymerase. 32P-products formed product was prepared with several enzymes; DNA with 3H-labeled single-stranded DNA were polymerase I from E. coli, AMV, and RLV isolated by CsCl gradient centrifugation and DNA polymerases (see above). After alkaline then subjected to HCHO denaturation as de- hydrolysis, carrier 2' (3') -mononucleotides were scribed above. Under these conditions, quantita- added and separated by high-voltage elec- tive separation of RNA and DNA was achieved. trophoresis. The area of the electrophoregrams In these experiments, the average chain of RNA corresponding to 2'(3') UMP contained 86, 90, was approximately 400 nucleotides.) The shift in and 92(%,' respectively, of 2p label in the mono- density observed in formaldehyde-heated prepara- nucleotide regions (Table 9). Further substantia- tions is most likely due to the single-strand breaks tion that the 32P-product present in these regions present in the 60 to 70S AMV RNA primer which contained 2' (3') phosphomonoester structure results in products containing a higher proportion was obtained; all of the 32p was converted to a of DNA. Norite-nonadsorbable form with alkaline phos- The covalent linkage of the DNA product to phatase. In contrast, 5' nucleotidase (crude snake the RNA is not limited to the viral RNA tem- venom) did not affect the Norite-adsorbable plate. Similar observations were made with the property of the 32P-product. The radioactivity VOL. 9, 1972 RNA-DEPENDENT DNA POLYMERASE. II 139

N 0 w0 x 4 E

.4 0) z 0r lr 09 I lto

FRACTION NUMBER FIG. 6. Gradienit cenitrifugationz oJ DNA polymerase product formed with f2 RNA as primer. (A) A DNA polymerase product was prepared in 0.1 ml with 76 oimoles of 3H-labeledf2 RNA (338 counts per miii per nimole), a32P-dTTP (5,000 countts per miii per pmole), 0.5 m.f MnCl2, aiid 0.28 uiiit ofpurified polymerase pII as described iin the text. After 15 miii of inicubationt at 38 C, the reactioni mixture was broughtt to a filnal conicenitrationl of 0.1 .if EDTA aiid 0.4% SDS in a volume of 0.25 ml; layered onito a 5-ml 5 to 20% linear sucrose gradieiit in 0.2% SDS, 20 mM sodium phosphate (pH 7), 1 mm ED TA, aiid 0.1 m NaCl; aiid cenitrifuged at 180,000 X g for 80 miii at 20 C in a Spiiico S W65 rotor. Thirty-drop fractions were collected by gravity after pierciiig the bottom of the centrifuge tube, precipitated with 5%( trichloroacetic acid, collected oni glass fiber filters, aiid counlited ill a sciiitillationi couniter. Uiitreated tritium-labeledf2 RNA (305) sedimeiited in a separate gradieiit at the same rate as the treated f2 RNA. (B) A DNA polymerase product prepared as above was heated to 65 Cfor 15 mill ill 3%70 fJrmaldehyde after additioii of ED TA aiid SDS. The product was anialyzed as above except that the sucrose gradieiit coiitaiiied 3%l, formaldehyde aiid was centtrifuged at 300,000 X g for 240 miii. The fbrmaldehlyde-treated f2 RNA sedimeiited with a sedimeiitatioii coefficient of 13S. Symbols: (0) 3H-f2 RNA; (@), 32P-dTMP-labeled produict. The ratio of 32p to 3H for each experimeiit is summarized in the iiiserls in the above figures. tivities primed by these different templates were 2 4 always found in the same fractions during enzyme E purification and were inactivated by heat at the w~~~~ same rate, suggesting that one enzyme is capable of using these different templates for DNA o 2 synthesis. A similar conclusion has been reached by Duesberg et al. (7). The incorporation of 0 5 0 5/ 20 2 w~ ~~FATO NI\\UMBE deoxynucleotides occurred at approximately the same rate with an equal amount of either DNA or RNA as template with the AMV polymerase. Furthermore, AMV polymerase primed deoxynucleotide incorporation with DNA or RNA FIG. 7. Equilibrium Cs2SO4 solutioii gradieiit ceii- was identically sensitive to a variety of trifiigatioii of DNA polymerase product prepared with templates poly (A)*poly (U). DNA polymeralse product labeled inhibitors. [Thymus DNA (nicked and treated with 2P-dTMP, 1heated at 75 C f2r 10 miii with 0.25 M with exonuclease III)-stimulated deoxynucleotide formaldehyde, aiid sedimeiited to equilibiruim ill Cs2SO4 incorporation was inhibited 51 % by anthramycin solutioii as described iii text. Symbols: (0), a32P-dTMP- (0.13 mM), 98%c by aurin tricarboxylic acid labeled produict; (0), density. (0.34 mM), 90%! by lucanthone (0.19 mM), and 99 by Congo red (0.12 mM), whereas camp- detected in the Ap region was not further char- tothecin (0.1 mM) had no effect. AMV RNA- acterized and may represent aberrant TMP stimulated deoxynucleotide incorporation was incorporation to the poly (A) chain. inhibited 46, 96, 73, 97, and 0, respectively, by the same inhibitors.] DISCUSSION The mechanism of deoxynucleotide incorpora- In the presence of four deoxynucleoside tion with RNA primers appears to be the same triphosphates and Mg2-, the purified viral as that found with DNA primers (14). With enzymes catalyze repair-like reactions on RNA, DNA primers, as well as synthetic RNA DNA DNA, or RNA DNA hybrids. Polymerase ac- hybrids, evidence had been presented that 140 LEIS AND HURWITZ J. VIROL. TABLE 9. Distributionz of 32p in mononucleotides after alkaline hydrolysisa 3

DNA polymerase II I I system used 2'(3') Mononucleotide II I I DNA polymerase AMV RuceRaviusher FIG. 8. Deoxynucleotide incorporation occurrintg at 3'-OH entd of primer stranids attached to template stranids. Arrow deniotes directioni of deoxyniucleotide Uridylate 86 90 92 inicorporaitiont. Guanylate <2 <2 <3 Adenylate 12 8 4 Cytidylate <2 <2 <2 As observed in reactions primed with DNA polymers, the polymerase does not initiate a Polymerase product was prepared in reactions synthesis of new chains de novo with RNA primed with poly (A)-poly (U) as described in primers but incorporates deoxynucleotides onto the text labeled with 5'a32P-deoxythymidine tri- the 3'-OH ends of the primer strands. Thus, the phosphate. The product was hydrolyzed with al- newly synthesized DNA is covalently linked to kali, and the mononucleotides were separated by RNA primer in a hybrid structure. This high-voltage electrophoresis in 0.05 M sodium the citrate (pH 3.5). The amount of 32p counted cor- attachment was demonstrated by the banding of responded to 4,400 counts/min (DNA polymer- AMV RNA-polymerase products in Cs2SO4 ase I), 450 counts/min [avian myeloblastosis solution in the hybrid density regions after virus (AMV) polymerase] and 1,120 counts/min treatment with formaldehyde. Similarly 32p_ (Rauscher virus polymerase). Control experiments dTMP-labeled products cosedimented with 3H-f2 were carried out with 32P-labeled poly (dT) syn- RNA primer-template in neutral sucrose-SDS thesized in reaction mixtures primed with poly gradients both before and after treatment with (A)-poly (dT). The product (1.09 X 105 counts/ formaldehyde. min) carried through the above procedure did not Only about 3% of the AMV RNA added as lead to the isolation of detectable nucleotides primer is transcribed into DNA. The size of the (<80 counts/min). in vitro synthesized DNA was determined to be deoxynucleotide incorporation occurs at the 3'- 6 to 7S by sedimentation in alkaline sucrose OH end of primer strands attached to template gradients, equivalent to a molecular weight of ap- strands. These structures must possess single- p roximately 1.7 X 105 to 2.6 X 105. DNA isolated stranded regions to direct synthesis as represented after incubation of partially permeable virus with in Fig. 8. Thus, when single-stranded regions of labeled deoxynucleoside triphosphates was found AMV RNA were degraded with Neurospora to have similar sedimentation coefficients (8, 9). nuclease, this RNA no longer supported deoxy- At present, it is not known whether the DNA nucleotide incorporation. As expected, the DNA synthesized in our system represents a hetero- synthesized with AMV RNA was largely re- geneous DNA population complementary to the sistant to the action of Neurospora and mung entire RNA genome as reported by Duesberg et bean nucleases, indicating its duplex structure. al. (9) for virion-generated DNA. In reactions primed with poly (A) -poly (U) (in Since viral RNA primers support a limited the presence of Mn24), the rate of dTMP in- extent of DNA synthesis, it appears that the corporation was markedly increased by the use DNA polymerase is unable to denature the of poly(U) previously nicked with micrococcal secondary structure of RNA. This limited nuclease followed by alkaline phosphatase treat- activity is similar to the activity found with the ment. Decreasing the poly(U) average chain T4-induced DNA polymerase and DNA poly- length from 200 to 300 nucleotides to 50 in- merase II of E. coli. With these two enzymes, creased the rate of dTMP incorporation 10- to specific proteins have been shown to play an 20-fold. Furthermore, the alkaline transfer experi- important role in repair reactions. The T4 gene ments (Table 9) indicate that the 3'-OH end of 32 protein specifically stimulates the T4-induced the poly(U) chain is the site of dTMP incorpora- DNA polymerase (1; B. Alberts et al., Fed. Proc. tion. 30:1036, 1971), and a protein recently isolated The DNA formed in the presence of AMV from E. coli specifically stimulates DNA poly- RNA is an RNA-DNA hybrid whose polymerase II in repair of DNA (M. Gefter and T. nucleotide content is mostly RNA. It bands in Kornberg, Fed. Proc. 30:1110, 1971). Similar CsCl and Cs2SO4 solutions at densities expected proteins may be present either in virions or in of products rich in RNA, and no labeled product infected cells and could facilitate viral polymerase was detected banding at a density of free DNA. transcription of RNA. VOL. 9, 1972 RNA-DEPENDENT DNA POLYMERASE. II 141

Manly et al. (16) and Fanshier et al. (11) re- ADDENDUM IN PROOF ported that both single- and double-stranded The work presented in this manuscript was pre- DNA free of RNA are produced by detergent- sented in preliminary form at the Federation Meetings treated virions. As of yet, we have not detected in June 1971. After this paper was submitted for publi- free DNA products in our purified system in, cation, evidence for covalent attachment of DNA vitro but rather only RNA-DNA hybrids. Thus products to RNA was reported by I. M. Verma, N. L. we believe that factors present in the virion Meuth, E. Bromfeld, K. F. Manly, and D. Baltimore, participate in the formation of free DNA. Possible Nature (London) 233:131. Also, the authors have mechanisms yielding free and double-stranded since learned that evidence of covalent attachment of DNA from RNA to DNA and has been obtained by P. Dues- RNA DNA hybrids might include berg and M. Bishop their respective co-workers (per- the following: (i) the covalent linkage between sonal communicationi). DNA and RNA could be hydrolyzed by an endonuclease in the virion. This cleavage would be followed by denaturation of the DNA chain LITERATURE CITED from the RNA by a concerted process involving 1. Alberts, B., and L. Frey. 1970. T4 bacteriophage gene 32: a structural protein in the replication and recombination of transcription and pealing as postulated by DNA. Nature (London) 227:1313-1318. Manly et al. (16). Since the viral polymerase does 2. Bader, J. P., and T. L. Steck. 1969. Analysis of the ribonu- not act at single-strand breaks (at least with cleic acid of murine leukemia virus. J. Virol. 4:454-459. DNA), this model requires localized denatura- 3. Baltimore, D. 1970. Viral RNA-dependent DNA polymerase. Nature (London) 226:1209-1211. tion which could be catalyzed by a T4-gene 4. Bray, G. A. 1960. A simple efficient liquid scintillator for 32-like protein. It is equally possible that both counting aqueous solutions in a liquid scintillation counter. mechanisms occur concomitantly. (ii) Inherent Anal. Biochem. 1:279-285. in the model of the primer-template role that viral 5. Duesberg, P. H. 1968. The RNA's of influenza virus. Proc. Nat. Acad. Sci. U.S.A. 59:930-937. RNA plays is the importance of hair-pin struc- 6. Duesberg, P. H. 1968. Physical properties of Rous sarcoma tures. If the polymerase can only elongate chains virus RNA. Proc. Nat. Acad. Sci. U.S.A. 60:1511-1518. in repair-like reactions, it would be unable to 7. Duesberg, P. K., K. V. D. Helm, and E. Canaani. 1971. Prop- transcribe the regions of the RNA involved in the erties of a soluble DNA polymerase isolated from Rous sarcoma virus. Proc. Nat. Acad. Sci. U.S.A. 68:747-751. hair-pin structure. This obvious loss of genetic 8. Duesberg, P. H., and W. S. Robinson. 1966. Nucleic acid and information can be obviated if covalently linked proteins isolated from the Rauscher mouse leukemia virus. DNA can form hair-pins intramolecularly. Proc. Nat. Acad. Sci. U.S.A. 55:219-227. Alternatively, in vivo initiation may involve other 9. Duesberg, P. H., P. K. Vogt, and E. Canaani. 1971. Structure and replication of avian tumor virus RNA, p. 154-166. In mechanisms. It is possible that small 3'OH- L. G. Silvestri (ed.), Lepetit Colloq. on Biol. Med., The terminated polynucleotides can serve as nuclea- Biol. of Oncogenic Viruses. North Holland Publishing Co., tion points for DNA synthesis, or additional Amsterdam. factors may operate to facilitate initiation of DNA 10. Erikson, R. L. 1969. Studies on the RNA from avian myelo- on viral RNA without the direct participation of blastosis virus. Virology 37:124-131. 11. Fanshier, L., A. C. Garapin, J. McDonnell, A. Faras, W. a primer strand. To date, however, no known Levinson, and J. M. Bishop. 1971. Deoxyribonucleic acid DNA polymerase can initiate new chains in polymerase associated with avian tumor viruses: secondary DNA-directed reactions. If DNA is an inter- structure of the deoxyribonucleic acid product. J. Virol. mediate in the replication of viral RNA it is 7:77-86. necessary to transcribe this DNA into RNA 12. Faras, A., L. Fanshier, A. Garapin, W. Levinson, and J. M. Bishop. 1971. Deoxyribonucleic acid polymerase of Rous progeny. Whether this transcription involves a sarcoma virus: studies on the mechanism of double stranded host cell or viral RNA polymerase is unknown. deoxyribonucleic acid synthesis. J. Virol. 7:539-548. At present, we as well as others (3, 24, 26) have 13. Green, M., M. Rokutanda, K. Fujinaga, R. K. Ray, H. not detected ribonucleotide incorporation with Rokutanda, and C. Gurgo. 1970. Mechanism of carcino- or crude extracts of virions under a wide genesis by RNA tumor viruses. I. An RNA-dependent virions DNA polymerase in murine sarcoma viruses. Proc. Nat. variety of conditions. It is evident that our under- Acad. Sci. U.S.A. 67:385-393. standing of the RNA oncogenic viruses would be 14. Hurwitz, J., and J. P. Leis. 1971. RNA-dependent DNA po- facilitated by the discovery of the mechanism of lymerase activity of RNA tumor viruses. I. Directing in- this RNA replication. fluence of DNA in the reaction. J. Virol. 9:116-129. 15. Johnson, P. H., and M. Laskowski. 1970. Mung bean nuclease ACKNOWLEDGMENTS 1. It. Resistance of double-stranded DNA and susceptibility We thank Pat Olenen for technical assistance in part of these of regions rich in adenosine and thymidine to enzymatic studies. hydrolysis. J. Biol. Chem. 245:891-898. This study was conducted under Public Health Service con- 16. Manly, K. F., D. F. Smoler, E. Bromfield, and D. Baltimore. tract 71-2251 within the Special Virus Cancer Program of the 1971. Forms of deoxyribonucleic acid produced by virions National Cancer Institute, and research grant from the National of the ribonucleic acid tumor viruses. J. Virol. 7:106-111. Institute of General Medical Sciences GM-13344 and by American 17. Mikulski, A. J., and M. Laskowski. 1970. Mung bean nu- Cancer Society grant P561-B. J. P. L. is a postdoctoral fellow clease I. III. Purification procedures and (3')-w-monophos- of the Damon Runyon Cancer Foundation. phatase activity. J. Biol. Chem. 245:5026-5031. 142 LEIS AND HURWITZ J. VIROL.

18. Monod, J., J. P. Changeux, and J. Jacob. 1963. Allosteric DNA hybrid molecules by the DNA polymerase of sarcoma- proteins and cellular control systems. J. Mol. Biol. 6:306- leukemia viruses. Nature (London) 227:1026-1028. 329. 23. Scolnick, E., E. Rands, S. A. Aaronson, and G. J. Todaro. 19. Rabin, E. Z., M. Mustard, and M. J. Fraiser. 1968. Specific 1970. RNA dependent DNA polymeraiise activity in five inhibition by ATP and other properties of an endonuclease RNA viruses; divalent cation requir-ements. Proc. Nat. of Neurospora crossa. Can. J. Biochem. 46:1285-1291. Acad. Sci. U.S.A. 67:1789-1796. 20. Robinson, W. S., and M. A. Baluda. 1965. The nucleic acid 24. Spiegelman, S., A. Burny, M. R. Das, J. Keydar, J. Schlom, from avian myeloblastosis virus compared with the RNA M. Travnicek, and K. Watson. 1970. Chalracterization of from the Bryan strain of Rous sarcomiia virus. Proc. Nat. the pr-oduct of RNA directed DNA polymerase in onco- Acad. Sci. U.S.A. 54:1686-1692. genic RNA viruses. Nature (London) 227:563-567. 21. Robinson, W. S., A. Pitkamen, and H. Rubini. 1965. The 25. Spiegelman, S., A. Bur-ny, M. R. Das, J. Keydar, J. Schlomli, M. Tr-avnicek, and K. Watson. 1970. DNA-directed DNA nucleic acid of the Br-yan striain of Rous sarcomla vir'us: polyrnerase activity in oncogenic RNA viruses. Naiture purification of the vir-us and isolationi of the nucleic acid. (London) 227:1029-103 1. Proc. Nat. Acad. Sci. U.S.A. 54:137-144. 26. Terniin, H. M., and S. Mizutani. 1970. RNA-dependent DNA 22. Rokutanda, M., H. Rokutanda, M. Gr-een, K. Fujinalga, polymerase in virions of Rous sarcoma virus. Nature (Lon- R. K. Ray, and C. Gurgo. 1970. Formai.ltioni of viral RNA- don) 226:1211-1213.