Repriated fror: Biochimica et Biophysica Acta Elsevier Publishing Company. Amsterdam - Printed in The Netherlands BBA 946997 PURIFICATION OF THE DNA POLYMERASE OF AVIAN MYELOBLASTOSIS VIRUS D.L. KACIAN, K. F. WATSON, A. BURNY anv S. SPIEGELMAN instituie of Cancer Research, College of Paysicians and Surgeons, Columbia University, New York, ALY. 10032 (U.S.A.5 ‘ (Received June rst. 1971} SUMMARY DNA polymerase from avian myeloblastosis virus has been purified by a com- bination of column chromatography and gel filtration methods. The isolated enzyme sediments at approximately 6S and consists of two subunits of molecular weights 10 000 and 69 ooo. It is free of RNA and DNA endonuclease activity. The enzyme possesses the RNA-, DNA-, and hybrid-directed polymerase activities found in the virion. INTRODUCTION The discovery)? of a ribonuclease-sensitive DNA poivmerase activity in on- cogenic RNA viruses was quickly extended*~’ to a wide variety of oncornaviruses®, It was farther shown that the product DNA was complementary to the RNA of the virion used as the source of the enzvme preparation® ®-4_ These findings were promptly followed by experiments that established the existence in these viruses of polymerase activities that respond to doubie-stranded DNA®12-18 and, with a very high effi- ciency. to synthetic homopolymeric dupiexes!*16 composed of polyribonucleotides polydeoxyribonucleotides, and hybrid structures of the two. The size of the DNA product synthesized was generally much less than that of the template employed? 4% 11, In addition to the DNA polymerase activities, evidence was also found for DNA endo- and exonucleases!®?7, ligase!”, and a nucleosidetriphosphate phospho- transferase in the virion Puritication of the RNA dependent DNA polymerase is a necessary prerequi- site to an unambiguous analysis of the reaction mechanism. Purity is also requi- red to delineate the relation of this polymerase activity to the others observed with DNA and synthetic homopolymeric duplexes as templates. Finally, pure enzyme should permit a decision on whether the nuclease, ligase and phosphotransferase ac- tivities are collectively or individually inherent and necessary components of the DNA polymerase function. We report here the purification and characterization of the DNA polymerase activity from avian myeloblastosis virus. Abbreviations: AMV, avian myeloblastosis virus; BBOT, 2,5-bis-2-(5-¢ert.-butylbenzoxa- zolyl)thiophene. Biochim. Biophys. Acta, 246 (1971) 365-383 366 D. L. KACIAN et al. MATERIALS Whatman microgranular DEAE-cellulose, DE52, 1.0 mequiv/g dry weight, and phosphocellulose, P11, 7.4 mequiv/g dry weight were obtained from Reeve Angel. Sephadex G-200 and CM-Sephadex C-50 were purchased from Pharmacia Fine Che- micals, Clarkson Chemical Company provided hydroxylapatite. Unlabeled nucleoside triphosphates and dithiothreitol came from P-L Biochemicals. Miles Laboratories supplied polynucleotides and Micrococcus lysodetkticus DNA. Tritiated nucleoside triphosphates were obtained from New England Nuclear, Schwarz BioResearch, and Amersham-Searle. Nucleic acid polymers were the generous gifts of Drs. F. Bollum (University of Kentucky), A. N. Nussbaum (Hoffman-LaRoche), and L. A. Under- kofler (Miles Laboratories). Acrylamide and methylene bisacrylamide came from Bio-Rad Laboratories. Nonidet P-40 was a product of Shell Chemical Co. METHODS (1) Purification of avian myeloblastosts virus Avian myeloblastosis virus (AMV), BAT strain A, was obtained by methods previously described!® from the blood of chicks in the terminal stage of myeloblastic leukemia”® and from infected myeloblasts suspended in tissue culture. Virus from blood plasma was purified essentially as described by CARNEGIE et al.4, As a final step, the virus suspension was sedimented at 27 000 rev./min in the Spinco SW27 rotor through 12 ml of 20 % glycerol in 0.01 M Tris-HCI (pH 8.5), 0.15 M NaCl, 1 mM EDTA (Tris-NaCl-EDTA buffer) onto a 6-ml pad of glycerol. The virus was removed from the pad, suspended in the same buffer without glycerol, and stored at —70°. Myeloblastosis virus produced in tissue culture was supplied by Dr. J. W. Beard, Duke University. The culture fluid had been concentrated 50-fold by centrifugation and contained from 2.5 - Io! to 5 - 101# virus particles per ml”. After centrifugation at 3000 xg for 10 min, the virus was concentrated against a 6-ml pad of glycerol at 27 000 rev./min for I h in the Spinco SW 27 rotor. Further purification was as pre- viously described®. (2) Isolation of AMV RNA Purified virus from blood plasma was lysed by adding sodium dodecyl sulfate to 0.5 %. The suspension was extracted twice with phenol-cresol solution (prepared according to KrrBy?’ and equilibrated with Tris-NaClI-EDTA buffer) and the RNA was precipitated by addition of 0.1 vol. of 3 M NaCl and 2 vol. of 95 % ethanol. After a second alcohol precipitation, the RNA was layered onto a Io to 30 % glycerol gradient containing 0.ox M Tris-HCl (pH 7.4), 0.1 M NaCl, and 1 mM EDTA in the Spinco SW 41 rotor. After centrifugation at 41 000 rev./min and 5° for 3 h, fractions were collected dropwise from the bottom of the tube and those containing the 70-S RNA component were pooled and alcohol precipitated. (3) Preparation of polynucleotide duplexes Polynucleotide duplexes were formed by annealing equimolar amounts of two complementary homopolymers at concentrations of approximately 100 ug/ml each in o.or M Tris-HCl (pH 7.4), 0.2 M NaCl at room temperature for 15 min. Biochim. Biophys. Acta, 246 (1971) 365-383 PURIFICATION OF AMV DNA POLYMERASE 367 (4) Polyacrylamide gel electrophoresis of proteins Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed by a modification of the method of SHaprro et ai.4. Protein samples were precipitated with an equal volume of 10 % trichloroacetic acid, allowed to stand at 0° for 15 min, and centrifuged at 16 000g for 30 min. Recovery was greater than 95 %. The pellet was thoroughly drained, and the pre- cipitated protein was dissolved in 25-50 ul of 0.01 M sodium phosphate (pH 7.8), 1 % sodium dodecyl sulfate, 1% 2-mercaptoethanol. After 30 min at 60°, glycerol was added to 10 % and the sample was layered onto the gel. Gels contained 5 % acrylamide, 0.25 % methylene bisacrylamide, 0.1 % sodium dodecyl sulfate, and 0.1 M sodium phosphate (pH 7.8). Electrophoresis was performed at 10 mA per gel for 15 min and then at 15 mA per gel for 75 min. Gels were stained for 2h in a 0.25 % solution of Coomassie brilliant blue in Io % acetic acid, 50 % methanol. They were destained by diffusion in 7 % acetic acid, 5 % methanol and stored in the same solvent. Molecuiar weights of polypeptide chains were determined as described by Wot et al. using as molecular weight markers polymers of ribonuclease A prepared ith diethyl pyrocarbonate. Electrophoresis of proteins at pH 8.9 in Tris-glycine buffer was performed as described by Davis?®. (5) Protein determination Protein was measured by the method of Lowry ef al.?” using crystalline bovine albumin (Fraction V) as standard. (6) Polymerase assay The assay mixture for homopolymer templated reactions (total volume 0.1 ml) contained the following in wmoles: Tris-HCl (pH 8.3), 5.0; MgCl,, 0.6; 0.02 each of the required labeled and unlabeled deoxynucleoside triphosphates; and double-stran- ded homopolymer template, 1.2 +103 pmoles polymer phosphate in each strand. Reactions were incubated at 37° for 10 min and terminated by the addition of coid 5 % trichloroacetic acid. After 10 min, the acid-precipitable radioactivity was collected on nitrocellu- lose filters and counted in 0.4 % 2,5-bis-2- (5-¢ert.-butylbenzoxazolyl)thiophene(BBOT) in toluene. Assays using natural RNA and DNA templates were prepared identically ex- cept that they contained 0.02 umoles each of three unlabeled nucleoside triphos- phates and 4nmoles of the fourth labeled triphosphate. Templates were used at levels from I to 2 ug per 0.x ml assay. Specific activities of the 3H-labeled triphosphates were 35-50 counts/min per pmole for homopolymer-templated reactions and 350-500 counts/min per pmole for those using natural RNA or DNA templates. (7) Preparation of AMV DNA polymerase The procedure is described for 60 mg of purified viral protein. Larger amounts have been handled successfully by scaling up the various steps proportionately. 1zml of AMV (5 mg/ml in 0.01 M Tris-HCl (pH 8.5), 0.15 M NaCl, 1mM EDTA) were mixed in order with 1.2 ml Nonidet P-40, 1.2 ml 10 % sodium deoxy- Biochim. Biophys. Acta, 246 (1971) 365-383 368 D. L. KACIAN ef al. cholate, and 3.6m! 4M KCl until homogeneous. The mixture was kept at 0° for 15 min and then was centrifuged at 16 000 xg for 10 min. The pellet was discarded and the supernatant diluted to 10 times its volume with 0.01 M potassium phosphate (pH 7.2), 2 mM dithiothreitol, 10 % glycerol. The solution was applied to a 1.2cmX1I.0cm column of DEAE-cellulose carefully equilibrated with the same buffer. The column was washed with 80 ml of 0.05 M potassium phosphate (pH 7.2), 2 mM dithiothreitol, 10 % glycerol, and eluted with 4o ml 0.3 M potassium phosphate (pH 7.2), 2 mM dithiothreitol, 10 % glycerol. The flow rate was about 36 ml/h. The peak activity fractions from the DEAE-cellulose column were pooled and diluted to 3 times their volume with 0.01 M potassium phosphate (pH 8.0), 2 mM dithiothreitol, 10 % glycerol. The material was loaded onto a 0.9 cm x 8.0 cm column of CM-Sephadex C-50 previously equilibrated with the same buffer. The column was washed with 8ml of 0.1 M potassium phosphate (pH 8.0), 2 mM _ dithiothreitol, 10 % glycerol and eluted with 12 ml of 0.3 M potassium phosphate (pH 8.0), 2 mM dithiothreitol, 10 % glycerol. A flow rate of 15 ml/h was maintained. The peak fractions were pooled, glycerol was added to 50 %, and the enzyme stored at —20°. (8) Phosphocellulose column chromatography of AMV DNA polymerase The peak fractions from a DEAE-cellulose column (about 5 mg protein) were pooled and diluted 6-fold with o.or M potassium phosphate (pH 8.0), 2 mM dithio- threitol, 10 % glycerol and applied to a 0.9 cm Xx g.0 cm column of phosphocellulose equilibrated with the same buffer. The column was eluted with a 150-ml gradient from 0.05 M potassium phos- phate (pH 8.0) to 0.5 M potassium phosphate (pH 8.0) containing 2mM dithio- threitol and 10 % glycerol. The flow rate was maintained at about 0.4 ml/min and about 1.5-ml fractions were collected. (9) Hydroxylapatite column chromatography of AMV DNA polymerase The peak fractions from a phosphocellulose column were pooled and diluted 5-fold with o.or M potassium phosphate (pH 7.2), 2 mM dithiothreitol, 10 % glycerol and loaded onto a 0.9 cm Xg.0 cm column of hydroxylapatite equilibrated with the same buffer. The column was eluted with a 150-ml gradient from 0.05 to 0.5 M potassium phosphate (pH 7.2) containing 2 mM dithiothreitol and 10 % glycerol. The flow rate was maintained at about 0.2 ml/min and about 2.0-ml fractions were collected. (10) DNA cellulose chromatography of AMV DNA polymerase DNA cellulose was prepared essentially as described by ALBERTS AND HER- RICK?8, Clean cellulose (Munktell 410) was washed several times with boiling ethanol and distilled water to remove remaining pyridine. It was then pre-cycled with base and acid (0.1 M NaOH, water, o.or M HCl) and washed to neutrality with water. The cellulose was then thoroughly dried, first in air and then by lyophilization. Calf thymus DNA was dissolved in 0.01 M Tris-HCl (pH 7.4), 1mM EDTA at a concentration of x mg/ml. The DNA solution was poured into petri dishes and mixed with the cellulose to form a slurry (approximately 1 g cellulose to 3 ml DNA solution). The material was extensively air dried, ground to a powder, and lyophi- Biochim. Biophys. Acta, 246 (1971) 365-383 PURIFICATION OF AMV DNA POLYMERASE 369 lized. Slow, complete drying seems to be essential for good adsorption of the DNA. The powder was resuspended in 0.01 M Tris-HCl (pH 7.4},1 mM EDTA, 0.15 M NaCl, washed twice with the same buffer, and checked for DNA adsorption by measuring optically the amount of DNA released by boiling. About 30-40 °% of the input DNA was taken up by the cellulose For chromatography of AMV DNA polymerase, a 0.5 cm 10cm column of DNA cellulose was exhaustively equilibrated with 0.01 M potassium phosphate (pH 8.0}, 2 mM dithiothreitol, 10° glycerol. Approximately. 60 ug of AMV DNA polymerase (phosphocellulose fraction) was applied to the column in about 0.03 M potassium phosphate buffer. The column was eluted with a 32-ml linear gradient from 0.01 to 0.5 M potassium phosphate (pH 8.0) containing 2 mM dithiothreitol, 10 % glycerol. The flow rate was maintained at 8 ml/h and 0.5 ml! fractions were collected. (ZI) Glycerol gradient centrifugation of AMV DNA polymerase AMV DNA polymerase (phosphocellulose fraction, approximately 0.7 mg) was layered over a Io to 30 % (v/v) glycerol gradient in 0.2 M potassium phosphate (pH 8.0}, 2 mM dithiothreitol in the Spinco SW 50.1 rotor. Bovine serum albumin was run on a parallel gradient to serve as marker. The proteins were sedimented at 50 000 rev. jmin and r° for 9.5 h and 1o-drop fractions were collected dropwise from the bottoms of the tubes through a 20-G needle. (72) Assay of contaminating nuciease activities Ribonuclease activity in the CM-Sephadex enzyme was measured by following the breakdown of *H-iabeled Escherichia coli 4-S and 5-S RNA on polyacrylamide gels. The RNA (15 wg) and enzyme (0.35 wg) were incubated in 0.025 ml of the standard assay mixture lacking deoxyriboside triphosphates and template. After 0 min and 60 min of incubation at 37°, sodium dodecy! sulfate was added to 1 %, and the samples subjected to electrophoresis on polyacrylamide gels as described by Bisuop et al.**, The gels were frozen, cut into 1-mm slices, dried on filter paper strips and counted in 0.4 °% BBOT in toluene. 7 Deoxyribonuclease activity was measured by following the breakdown of #H- labeled E. coli DNA by alkaline sucrose gradient centrifugation. - Two standard reaction mixtures were prepared omitting the deoxyribonucleo- tides and including the labeled DNA (approximately 0.30 Bg). Purified AMV DNA polymerase (1.4 ug) was added to one, and both were incubated at 37°. After 30 min, EDTA was added to 5 mM and sodium dodecyl sulfate to 0.5 %, and the samples were layered onto 5 to 20 °% sucrose gradients containing 0.1 M NaOH, 0.9 M NaCl, and 1mM EDTA in the Spinco SW 50.1 rotor. After centrifugation at 50 000 rev./ min for 3h at ro°, fractions were collected dropwise from the bottom of the tube and precipitated with trichloroacetic acid. Insoiuble material was collected on nitro- cellulose filters and counted in BBOT-toluene scintillation fluid. RESULTS Extraction of the DNA polymerase from virions Solubilization of the AMV DNA polymerase was effected by treatment of the Biochim. Biophys. Acta, 246 (1971) 365-383 370 D. L. KACIAN éf al. virus particles with detergent (0.7% deoxycholate; 7% Nonidet P-4o) and salt (0.8 M KCl) at 0°. Glycerol gradient analysis in 0.1 M potassium phosphate showed that over 95 % of the enzyme activity sedimented at 8.4 S or less after treatment. Assays were performed using poly (rA)- poly (U), AMV 70-S RNA, and M. lyso- detkticus DNA as templates. Specific activities and recoveries were determined with poly (rA)+ poly (rU). Lower concentrations of detergent or salt, while capable of releasing the enzyme from the virion, left much of the activity attached to material that sedimented at higher s values. After release the enzyme showed an absolute requirement for added template. The extract was centrifuged at low speed to remove a small amount of material that reduced the flow rate of the DEAE-cellulose column. The pellet contained a negligible amount (< 3 %) of the activity. Chromatography of AMV DNA polymerase on DEA E-cellulose The solubilized enzyme was diluted ro-fold to reduce the concentrations of salt and detergent and loaded onto a column of DEAE-cellulose. The column was then exhaustively washed with 0.05 M potassium phosphate buffer, which removes the detergents and much of the protein. Virtually all the activity was retained through- out loading and washing. The enzyme was then eluted with 0.3 M potassium phos- phate buffer. About 5 % of the protein is recovered from the column, together with most of the activity. Fig. 1 shows a DEAE-cellulose column assayed, respectively, with three diffe- rent templates: the homopolymer duplex poly (rA)- poly (rU), 70-S AMV RNA, and M. lysodetkticus DNA. With each template, the activity is found generally to coincide with the protein peak. The slight displacement of activity observed with various templates appears to be due to their differential sensitivity to contaminating proteins and salts. Generally, greater than 90 % of the starting activity is eluted with the 0.3 M potassium phosphate. The DEAE-cellulose column step yields about a 20-fold en- richment of the enzyme. CM-Sephadex chromatography of AMV DNA polymerase The peak activity fractions from the DEAE-cellulose column were pooled, diluted 3-fold with low salt buffer, and loaded onto a column of CM-Sephadex. The column was washed with o.1 M potassium phosphate and eluted with 0.3 M potas- sium phosphate. All of the enzyme activity is retained by the column during loading and washing. Fig. 2 shows the profiles obtained by assaying the column with three different templates. About 0.5-1 % of the total protein is eluted with the 0.3 M buffer. The degree of purification after CM-Sephadex chromatography and the amount of activity recovered have varied with different batches of virus, but highly reprodu- cible results are obtained when the same starting material is used. In every case, the column effectively removes all the acidic protein contaminants as measured by polyacrylamide gel electrophoresis at pH 8.9. Preparations using different batches of virus produced in tissue culture have yielded enzyme of 30—-60-fold higher specific activity than the crude extract. Variability in yield is probably due to the instability of the enzyme. The history of the virus preparation may be of paramount importance Biochim. Biophys. Acta, 246 (1971) 365-383 PURIFICATION OF AMV DNA POLYMERASE 371 in obtaining high yields and specific activities. It appears that certain treatments (e.g. multiple freezing and thawing) adversely affect the stability of the enzyme. An actual enzyme preparation carried through the CM-Sephadex step is sum- marized in Table £. The CM-Sephadex enzyme was assayed as described in METHODS for RNA and DNA endonuclease activities. As can be seen in Figs. 3 and 4, no detectable break- a 2, poly (rAj-poly (rus i " ul pmoles x!0% ['H] dTMP incorporated (1a / 6u2) wieioug (1037 Bus) wyeyorg pmoles Pu dTMP incorporated 4r 4 DNA (M. lysod.). (tw/ Bu) uisjorg pmoles [°H] dTMP incorporated Fig. 1. DEAE-cellulose chromatography of AMV DNA polymerase. Avian myeloblastosis virus (600 mg) was solubilized as described in METHODS and chromatographed on a 2.5 cm x25 cm column, Fractions of 4.4 ml were collected from the 0.3 M phosphate eluent. Assays using poly (rA) + poly (rU), AMV 70-S RNA, and M. lysodeikticus DNA were performed using 2 yl, 5 ul and 5 wl, respectively, from each fraction. Biochim, Biophys. Acta, 246 (1971) 365-383 372 D. L. KACIAN et al. down of the nucleic acids occurred when incubated with purified enzyme under stand- ard conditions, minus the deoxyriboside triphosphates. Enzyme stored in 50 % glycerol at —20° has retained greater than 90 % of its activity for more than 5 weeks. wi v ky poly(rA)- poly (ru) Nw pmoles x10" [x] GTMP incorporated pmoles [?H] dTMP incorporated pmoles [°H] dTMP incorporated 10 20 3% 40 Fraction Fig. 2. CM-Sephadex chromatography of AMV DNA polymerase. The peak fractions (19-24) of the DEAE-cellulose column shown in Fig. 1 were pooled and chromatographed on a 1.5 cm > 17.5 cm column. Fractions of 1.5 ml were collected from the 0.3 M phosphate eluent. I Ha, 5 Hl, and 5 ul were used, respectively, to assay activity with poly (rA)- poly (rU), AMV RNA, and DNA. Biochim. Biophys. Acta, 246 (1971) 365-383 PURIFICATION OF AMV DNA POLYMERASE 373 TABLE I PURIFICATION OF DNA POLYMERASE FROM AVIAN MYELOBLASTOSIS VIRUS Assayed in the standard assay mixture using poly (rA) - poly (rU) as template. Specific activity expressed as pmoles dTMP incorporated per 10 min per yg of protein. Fraction Total Specific Total Yield protein activity activity (%) (mg) (units |ug) (units) (1) Solubilized virus 155-4 26.4 4.1 + ros 100 (2} DEAE-cellulose column pool 8.0 487.5 3.9 + 108 95 (3) CM-Sephadex pool 1.5 909.0 I.4° 108 34 ar | wee min < i 7 : oo DNA POLYMERASE é | io NO DNA POLYMERASE & 7 1 5 6 h vA eo | | & ‘ x 4 ‘ & ab : 3 : bE | ° g a 43 5 24 Bt | s & ale 29 2 a | | 4,60 min 2 5 2 ivy i = | , OT : fs “tines I ynpsnntets Stee, 10 20 30 40 50 60 5 10 5 20 25 DISTANCE (mm) FRACTION Fig. 3. Assay of ribonuclease activity in CM-Sephadex enzyme. *H-labeled E. coli 4-S and 5-S RNA (30 000 disint./min per ug) were incubated with CM-Sephadex enzyme as described in METHODS and resolved on 4.8 % pre-swollen polyacrylamide gels at 10 mA per gel for 60 min. Fig. 4. Assay of deoxyribonuclease activity in CM-Sephadex enzyme. *H-labeled E. coli DNA (69 ooo disint./min per zg) was incubated with CM-Sephadex enzyme as described in METHODS and examined by alkaline sucrose density gradient centrifugation. Evidence for two components in the AMV DNA polymerase Peak fractions from several columns were pooled, denatured with sodium dodecyl sulfate and mercaptoethanol and analyzed on 5 % sodium dodecyl sulfate— polyacrylamide gels. Fig. 5 shows the band patterns at various stages of purification. After DEAE cellulose chromatography there are still over 25 different polypeptide chains present. The second gel reveals that after CM-Sephadex two bands become very prominent suggesting that they may be responsible for enzyme activity. This is further supported by the third gel of material passed through a Sephadex G-200 column. Fig. 6 shows the activity profile from the Sephadex G-z200 column and shows that the enzyme is separated from smaller ultraviolet-absorbing material. It is of interest. to note (Table II) that the Sephadex G-200 protein, which shows only two bands, retains the ability to respond to all the templates that were active with the crude detergent-disrupted virion preparations. Too much weight should not be given to the relative responses to the synthetic templates since they vary from one polymer preparation to another. Biochim. Biophys. Acta, 246 (1971) 365-383 374 D. L. KACIAN et al. 20, &2 poly (dT) + poly (rA) 7° > Yr eed l pmoles xl07 Pl dTMP incorporated O8ty Fraction RNA (AMV) DNA (M. lysod.) pmoles PH] dTMP incorporated 20 40 60 80 Fraction Fig. 5. Sodium dodecyl] sulfate-polyacrylamide gels of DEAE-cellulose, CM-Sephadex and Se- phadex G-200 enzyme fractions. Sodium dodecyl sulfate gels of AMV DNA polymerase were run after purification by DEAE-cellulose chromatography (left), CM-Sephadex chromatography (middle), and Sephadex G-200 chromatography. They contain, respectively, 32, 30, and 28 yg of protein. Fig. 6. Sephadex G-200 chromatography of AMV DNA polymerase. The peak fractions (475 #g protein in 1 mi) from a CM-Sephadex column were pooled and loaded onto a 0.9 cm x 54 cm column of Sephadex G-200 equilibrated with 0.3 M potassium phosphate (pH 8.0), 2 mM dithio- threitol, 5 % glycerol. The column was eluted at a flow rate of 7 ml/h and 0.6 ml fractions were collected. 10-l aliquots from each fraction were used to assay with each template. To gain further information on the association of the two principal bands with the polymerase activity, the behavior of the protein on other columns was examined. Biochim. Biophys. Acta, 246 (1971) 365-383 PURIFICATION OF AMV DNA POLYMERASE 375 TABLE II ACTIVITY OF PURIFIED AMV DNA POLYMERASE WITH VARIOUS TEMPLATES Assays were performed as described in METHODS except that all reactions were incubated at 37° for 10 min. Each contained 0.6 ug of Sephadex G-200 enzyme. {H]dTTP was used in all reactions except those with poly (dC) - poly (dG) and poly (rI) - poly (rC) where the label was in dGTP. Homopolymer duplex templated reactions contained 1.2+ 10% pmoles polymer phosphate in each strand. AMV RNA and M. lysodeikticus DNA were present at 1.2 ug and 2 ug per reaction, respectively. Template Labeled base incorporated (pmoles) Poly (dT) + poly (rA) 1294.6 AMV RNA 8.9 M. lysodeikticus DNA 7.6 Poly (dC) + poly (dG) 235.6 Poly (rA) + poly (rl) 440.5 Poly (rl) + poly (rC) 311.4 To this end, the DEAE-cellulose enzyme was put on phosphocellulose instead of CM-Sephadex and eluted with a phosphate gradient. As shown in Fig. 7, the enzyme activity elutes as a single sharp peak. The inset on Fig. 7 shows the sodium dodecyl sulfate gel analysis of the peak region and reveals the same two principal bands along with some minor contaminants. Fractions 32-39 of the active region from Fig. 7 were pooled and chromatographed on hydroxylapatite. The elution profile of the activity with a phosphate gradient is shown in Fig. 8 along with the sodium dodecyl sulfate gel of the peak region. Here we see the two principal bands as virtually the only detectable components. Enzyme eluted from another phosphocellulose column was then separated by affinity chromatography on DNA cellulose as described in METHODS. Once again (Fig. 9) the enzyme was recovered in a single sharp peak which, when analyzed by gel electrophoresis, was seen to contain the same two polypeptide chains. Ww Q © UD ~ 3 I 2 oO — w wa 2 carry 3 x 3 =z 5 200 Oo % ° 4 POly(dT) -poly (rA) = = b 8 UD a 3 - 6 8 = 100 weer 777058 8 & fo Fe eee 2 1 3 wo | flbaeer™ £ 2 ° a “ a =v 2 Sal Nafnatiaat aro | * 20 40 60 80 100 Fraction Fig. 7. Phosphocellulose column chromatography of AMV DNA polymerase. AMV DNA poly- merase (DEAE-cellulose fraction) was chromatographed on phosphocellulose as described in METHODS. 2 yl from each fraction were used to assay poly (AT) - poly {rA) templated activity and_-20 pl from each for AMV RNA activity. A sodium dodecyl sulfate-polyacrylamide gel of the peak fraction is shown. Biochim. Biophys. Acta, 246 (1971) 365-383 376 D. L. KACIAN et al. PURIFICATION OF AMV DNA POLYMERASE 377 Sedimentation of purified polymerase The two components appear to behave as a single complex on a variety of columns. It was of obvious interest to see whether this held true in a sedimentation analysis. A phosphocellulose enzyme preparation was accordingly run on a glycerol gradient containing 0.2 M potassium phosphate as described in METHODS. Fig. 10 shows that the activity sediments as a sharp band with an Sy9 ,, of 65 corresponding ' 50r- (e-a) payD4od4oqU dIALLP |He| Setowd pmoles BH dTMP incorporated (¢) Fraction Fig. 8. Hydroxylapatite column chromatography of AMV DNA polymerase. AMV DNA poly- merase (phosphocellulose fraction) was chromatographed on hydroxylapatite as described in METHODS. 10 ul from each fraction were used to assay poly (dT) - poly (rA) templated activity and 20 yl from each for for AMV RNA activity. A sodium dodecyl sulfate—-polyacrylamide gel of Fractions 26—37 is shown. Fractions 18-25 gave an identical but considerably lighter pattern. lo}- * poly(dC): poly(rG) ~ * JL ~ 4 er y 56 RNA (AMV) 19, 4r BSA a € Oo 4 6 3 g £9 2 2 uD x o ey - 3 o x BX 8 n 9 o 2 9 € EE © 2 © = T a © Ir —~ BE 2 L a 9 ¢. poly(dT)- ply (rA) 8 3 : 2 9 6 dos *v E o : ae a ¢£ oO * 20 40 & G6 a Qo . £ PS 4b 40.4 a Fraction 20 22 24 26 28 30 32 GU . aq 5 Y ES Fig. 10. Glycerol gradient centrifugation of AMV DNA polymerase. Centrifugation was per- 5 2 ok | DNA(Miysod) Igo formed as described in METHODS. Phosphocellulose enzyme was precipitated with an equal volume av . of saturated (NHy),SO, (4°, pH adjusted to 7.4 with NH,OH) suspended in o.1 ml of buffer and 33° layered onto the gradient. The activity was located using poly (dC) - poly (rG) as the assay tem- E Eo Olcieaoameeae plate and 10 yl from each fraction. The polymerase activity sedimented at approximately 6S calculated with reference to bovine serum albumin (BSA, arrow) run on a parallel gradient. FRACTION Fig. 11. Sodium dodecyl sulfate-polyacrylamide gels of glycerol gradient peak. The peak frac- Fig. 9. DNA cellulose chromatography of AMV DNA polymerase. AMV DNA polymerase (phos- tions 20, 22, 24, 26, 28, 30 and 32 from the glycerol gradient shown in Fig. 10 were each precipi- phocellulose fraction) was chromatographed on DNA cellulose as described in METHODS. 10 pl tated with trichloroacetic acid and analyzed by electrophoresis in sodium dodecyl! sulfate gels as from each fraction were used to assay for activity with each of the indicated templates. A sodium described in METHODS. The gels show that the polypeptide chains sediment together and that dodecyl sulfate-polyacrylamide gel of fractions 29-32 inclusive is shown. they coincide with the activity peak. The amount of protein recovered on each gel corresponds to the relative activity of the respective fractions. The arrow indicates the activity peak. Biochim. Biophys. Acta, 246 (1971) 365-383 Biochim. Biophys. Acta, 246 (1971) 365-383 378 D. L. KACIAN et al. to a molecular weight of r10 000. It may be noted that a similar sedimentation value has been reported®° for the DNA polymerase of the Rous sarcoma virus. The peak fractions (20-32 inclusive} were precipitated and analyzed electrophoretically in sodium dodecyi sulfate-acrylamide gels. As may be seen from Fig. 11, the same two bands are observed as the principal components in the peak region. It is evident that they are sufficiently tightly complexed to behave as a single physical entity under these conditions. Mole ratios and molecular weights of the two components The molecular weights were determined on sodium dodecy! sulfate-acrylamide gels using polymers of ribonuclease A as molecular weight markers. As may be seen from Fig. 12, the two chains have molecular weights of 110 000 and 69 000. If these two polypeptides are in fact subunits of the same enzyme, then the relative number of each present in an enzyme unit should be proportional to their 1 2 3 DISTANCE (cm) Fig. 12. Determination of molecular weights of subunits. The molecular weights of the polypep- tide chains present in AMV DNA polymerase extracts were determined on sodium dodecyl sul- fate-polyacrylamide gels as described in MeTHOoDs. The circles show the positions of polymers of ribonuclease A (mol. wt. 13 700) run as markers. The arrows mark the positions of the two major bands run on a parallel gel, indicating molecular weights of 110 000 and 69 ooo. Molecular weights of marker polymers range from 13 700 to 123 300. Fig. 13. Scan of sodium dodecyl sulfate-polyacrylamide gel of CM-Sephadex enzyme. Sodium dodecyl sulfate-polyacrylamide gels were scanned, using a 0.05 mm slit, in the Gilford Model 2400 spectrophotometer equipped with linear transport. The pattern shows that the enzyme is approximately 90 % pure assuming that all proteins are stained equally by Coomassie blue. Biochim. Biophys. Acta, 246 (1971) 365-383 PURIFICATION OF AMV DNA POLYMERASE 379 molecular weights. It has been shown®! that the amount of Coomassie brilliant blue bound to various proteins differed by less than 10 %. By scanning stained sodium dodecyl sulfate gels, the amount of each protein present can be determined3233. A typical scan is shown in Fig. 13 from which the ratio of the two components is readily obtained. Gels containing various amounts of enzyme from different sources were scanned and measured with the results shown in Table III. It is evident that the weight ratio of the chains found in the various preparations is that expected if the two components are present in equal numbers. TABLE IilI WEIGHT RATIO OF CHAINS Gels were scanned as described by BERG3?. The area under each peak was i i ; . estimated b Iti- plying the peak height by the peak width at half-height. P =_— Gel Enzyme preparation IIo 000 mol, wt. No. polypeptide : 69 000 mol. wt. polypeptide 34 DEAE-cellulose enzyme 1.3 53 CM-Sephadex enzyme 1.9 63 CM-Sephadex enzyme 1.7 64 Sephadex G-200 enzyme 1.5 67 CM-Sephadex enzyme 1.5 69 Sephadex G-200 enzyme 1.8 75 Phosphocellulose enzyme 1.4 83 Hydroxylapatite enzyme 1.3 88 Glycerol gradient enzyme 1.7 (Fraction 24, Fig. rr) 89 Glycerol gradient enzyme 1.6 (Fraction 26, Fig. 11) 90 Glycerol gradient enzyme 1.6 (Fraction 28, Fig. 11) Average of 11 gels: 1.6 Expected value (assuming one of each polypeptide chain per enzyme molecule): 1.6 TABLE IV REQUIREMENTS OF AMV DNA POLYMERASE REACTION Complete system: 50 mM Tris-HCl, pH 8.3; ; » Pp -3; 6mM MgCl,; 0.2 mM each dATP, dCTP, dGTP; p04 mM PHIdTTP, 665 counts/min per pmole; 0.7 4g AMV DNA polymerase, phosphocellu- ose fraction; 2ug AMV RNA; 0.4 mM dithiothreitol; roo mM KCl; incubated 20 min at 37°. System [8H] dTTP incorporated (pmoles) Complete 12.4 —MgCl, 0.0 —MgCi,+0.6 mM MnCl, 3.5 —dATP 0.4 —dCTP 0.3 —dGTP 0.5 —AMV RNA oO. ~dithiothreitol 8.6 —KCl 5-3 Biochim. Biophys. Acta, 246 (1971) 365-383 380 D. L. KACIAN et al. Properties and requirements of the reaction with purified AMV DNA polymerase The availabie information on the optimal conditions and requirements of RNA- dependent DNA polymerases have thus far been derived from studies with deter- gent-disrupted virion preparations. Table [V summarizes a reexamination with a purified AMV polymerase. It will be noted that the reaction is dependent on RNA, the presence of all four deoxyriboside triphosphates, and a divalent cation, Mg?+ being superior to Mn*+ over a wide range of concentrations. The addition of dithio- threitol and KCi lead to marked improvement. Fig. 14 shows that the effect of the dithiothreito! 1s most readily apparent in the later stages of prolonged syntheses. pmoles dTMP incorporated 50 100 ISO MINUTES Fig. 14. Effect of dithiothreitol on AMV DNA polymerase activity. Reaction kinetics in the presence (A) and absence (@) of 0.4 mM dithiothreitol (DTT) are shown. The template is AMV 7o-S RNA. The curves show that dithiothreitol is necessary for long-term synthesis. The exact time varies at which the reaction without dithiothreitol ceases. : DISCUSSION The two-column procedure described for preparing purified DNA polymerase from avian myeloblastosis virus is a rapid and convenient procedure for obtaining reasonably pure enzvme in good yield. At the CM-Sephadex stage, enzyme from tissue culture virus is go % pure or better. Substitution of phosphocellulose for CM-Sepha- dex chromatography gives similar results. If one starts with virus isolated trom the plasma of infected birds, enzyme oi even higher purity is obtained at corresponding stages of purification. Removai of residual contamination can be achieved by (NH,) SO, concentration foliowed by either Sephadex G-200 or hydroxylapatite chromato- graphy, or centrifugation through glycerol gradients. Two polypeptide chains are found to be associated with the activity in 1:1 ratio throughout purification by DEAE-cellulose, CM-Sephadex, Sephadex G-200, phosphocellulose, hydroxylapatite and DNA cellulose affinity chromatography as well as by glycerol gradient centrifugation. This suggests that they are subunits of the polymerase. However, proof that the two polypeptides are in fact subunits will require examination of the activities of the separated and reconstituted proteins. The possibility that the subunits may each have one or more of the activities associat- ed with them individually cannot be ruled out at this time. There is a 1.6-fold discrepancy between the molecular weight of the active form as determined by glycerol gradient centrifugation (110 000) and the sum of the molecular weights of the two polypeptide chains determined on sodium dodecyl sulfate-polyacrylamide gels (110 000 and 69000). Assuming that the molecular Biochim. Biophys. Acta, 246 (1971) 365~383 PURIFICATION OF AMV DNA POLYMERASE 381 weight determinations are not in error, the fact that the ratio of the chains is con- stant across the activity peak (Table III) in the glycerol gradient suggests that the discrepancy is due to differences in molecular asymmetry of the subunits as compared with assembled proteins. A quantitatively similar situation obtains with Qf repli- case in which the sum of the molecular weights of the subunits determined on so- dium dodecyl sulfate gels is 1.7 times that determined by sedimentation for the native enzyme 35, The value obtained for the sedimentation coefficient of the enzyme would seem to exclude the possibility that the molecule contains more than one of each subunit. It is worth emphasizing that association of the two major polypeptides, even through an extensive series of purification steps, does not constitute proof that they represent the desired enzyme. As with all such enzyme purifications, minor con- taminants (e.g. I % or less) that might not be detectable could conceivably be res- ponsible for the activity. We cannot prove rigorously at this time that the two prin- cipal polypeptide chains constantly observed in our active preparations compose the polymerase. However, in addition to their invariant presence and constant I : 1 ratio, several additional considerations argue for their being the enzyme components. Throughout purification, there have been no minor bands that are consistently present in all preparations. The most frequently observed contaminant is a pair of bands representing approximately 4 % of the total protein. From their gel mobilities, they have molecular weights of 190 000 and 210 000 and therefore would be expected to sediment much more rapidly, either individually or as a complex, than the fol- merase. They are not enriched relative to the two major bands during the course of purification of the activity. It seems likely that they represent aggregates of the major bands due to the relativelv large amount of protein applied to the gels. Were the activity due to any contaminant present as a few percent of the protein, the specific activity of the purified enzyme would be many-fold greater than that observed. The most active preparations of E. coli DNA polymerase assayed with poly [d(A-T)], Qf replicase?’ assayed with Q8 RNA, and E. colt transcriptase assayed with poly [d(A-T)]* and calf thymus DNA* have specific activities”, respectively, of 1.2, 3.2, 4.5, and 1.2 moles nucleoside monophosphate incorporated per sec per mole enzyme. The most active preparations of AMV DNA polymerase incor- porate 1.2 moles dXMP per sec per mole enzyme. This value is in excellent agreement with those for the other purified polymerases and argues that the principal protein components are responsible for activity. These calculations encompass both initiation and chain elongation. They are not based on any assumptions concerning the numbers of enzyme molecules participating, but rather on the reported enzyme specific activities. If in fact the enzyme is essentially pure, the amount of protein recovered should correlate with the number of enzyme molecules expected per virion. Itis reasonable to assume that there is at least one polymerase molecule per virus particle. Since the virion contains 3.0 - 10% daltons of protein*® and the molecular weight of the AMV DNA polymerase is 1.8- 105 daltons, one enzyme molecule per virion * The highest value obtained for enzyme fractions described as homogenous was used. The reported specific activities were converted from arbitrary units to moles of nucleoside mono- phosphate per sec per mole of enzyme. Biochim. Biophys. Acta, 246 (1971) 365-383 382 D. L. KACIAN é¢ al. would represent 0.06 % of the total protein. Normally, 0.3-1 % of the starting protein is recovered, which would correspond to 5-17 enzyme molecules per virus particle. Were the enzyme a 2 % contaminant of our preparation, each virion would contain only 0.1-0.3 polymerase molecule. 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