Biochem. J. (1973) 131, 237-246 237 Printed in Great Britain Multiple Forms of Nuclear Deoxyribonucleic Acid Polymerases and their Relationship with the Soluble Enzyme By R. L. P. ADAMS, MAUREEN A. L. HENDERSON, W. WOOD and J. G. LINDSAY Department ofBiochemistry, University of Glasgow, Glasgow G12 8QQ, U.K. (Received 31 July 1972) 1. DNA polymerase from nuclear and supernatant fractions of cultured mouse L929 cells was fractionated on columns of Sephadex G-200, Sepharose 4B and of DEAE- cellulose. Several peaks of activity are found on Sephadex chromatography and the distribution ofactivity between these depends on: (a) the source ofthe enzyme, i.e. nuclear or supernatant fraction; (b) the mode of extraction of the enzyme from the nucleus; (c) the amount of enzyme applied to the column. 2. The DNA polymerase activity in the lower-molecular-weight peaks (approximate molecular weights are 35000, 70000 and 140000) is firmly bound within the cell nucleus and shows a preference for native DNA as template, whereas the high-molecular-weight peak (peak I, molecular weight 250000 or greater) is found in supernatant fractions and shows greater activity with a denatured DNA template. 3. During periods of DNA synthesis the high-molecular-weight enzyme becomes more firmly bound within the nucleus. 4. Peak I enzymic activity is relatively unstable and is inhibited by thiol-blocking reagents and deoxycholate, but it is stimulated by univalent cations. 5. Very little endonuclease is present in the polymerase preparations, but a very active exonuclease and nucleoside diphosphokinase are present. On Sephadex chromatography, however, it was shown that the immediate precursors for DNA synthesis, at least by peak I enzyme, are the deoxyribonucleoside triphosphates. 6. Attempts to decrease the molecular weight of the peak I enzyme while still retaining activity failed. Several groups (Wallace et al., 1971; Haines et al., We have been unable to degrade the high- 1971; Weissbach et al., 1971) have now reported that molecular-weight enzyme to smaller active fragments, the DNA polymerase activity found in supernatant but have shown several important differences between fractions from animal cells is of higher molecular the high-molecular-weight and the lower-molecular- weight than the nuclear enzyme. However, no weight enzymes, in particular with regard to their evidence has appeared as to the function and com- relative stability, sensitivity to thiol-blocking reagents plexity of these enzymes nor as to their relationship and affinity for the DNA template. with each other. It is, however, known that the supernatant enzyme, which shows greater activity with a denatured DNA template, is more active in Materials and Methods whereas the nuclear enzyme, which growing tissues, Preparation of enzyme fractions normally prefers a native DNA template, shows considerable activity in cells and tissues not actively Mouse fibroblast (L929) cell cultures were sub- growing (Iwamura et al., 1968; Lindsay et al., 1970; cultured in minimum essential medium (Eagle) Ove et al., 1970). supplemented with 10% (v/v) of calf serum (medium We have confirmed that the supernatant enzyme is EC10). Cultures and media were obtained from larger than the nuclear enzyme and have shown that Flow Laboratories Ltd., Irvine, Ayrshire, U.K., or the latter contains at least three species of DNA from BioCult Laboratories, Glasgow, U.K. Cells, polymerase of approximate molecular weights 35000, which as a routine were tested for mycoplasma, were 70000 and 140000. harvested (a) while growing exponentially or (b) after Nuclei also contain DNA polymerase activity they had entered the stationary phase or (c) 2h after indistinguishable from the supernatant enzyme. There reversal of a 16h aminopterin blockade, i.e. in S- is increased association of the high-molecular-weight phase (Adams, 1969). Nuclear and supernatant enzyme with the nucleus during S-phase, and this fractions were prepared as previously described would account for the increased activity of S-phase (Lindsay et al., 1970) by homogenizing the washed nuclei with a denatured DNA template (Lindsay cells in 0.25M-sucrose buffered with 20mM-Tris- et al., 1970). HCI buffer, pH7.5. Vol. 131 238 R. L. P. ADAMS, M. A. L. HENDERSON, W. WOOD AND J. G. LINDSAY Table 1. Purification ofnuclear enzymes The values in parentheses represent the yields of enzymic activity. Sp. activity of DNA polymerase (units/mg) Total protein Native Denatured Fraction Sample (mg) template template I Whole nuclei 110 0.5 (100) 0.2 (100) II 0.4m-KCI extract 36 2.5 (160) 1.0 (167) III (NH4)2SO4 precipitate 19.4 5.8 (204) 3.9 (367) The homogenate was centrifuged at 800g for DNA polymerase assay 10min and the supernatant fraction, after being centrifuged at 105OO0g for 60min (Spinco model L DNA polymerase was assayed basically as de- ultracentrifuge, rotor no. 40), was used as a source scribed by Shepherd & Keir (1966). Samples (0.1 ml) of soluble enzyme. The nuclear pellet was washed in column buffer were incubated at 37°C for 60min in twice in buffered sucrose and was then extracted a total volume of 0.25ml with 4,umol of Tris-HCl with medium containing (final concentrations) 0.4M- buffer, pH7.5, 1.5,umol of MgCl2, 15,umol of KCI, KCl, 20mM-Tris-HCl buffer, pH7.5, and 5mM-2- 0.1,imol of EDTA, 3,umol of 2-mercaptoethanol, mercaptoethanol to yield the nuclear enzyme. 100l,g of DNA and 50nmol each of dATP, dGTP, Enzymic preparations were concentrated by adding dCTP and [Me-3H]dTTP (20nCi/nmol) (The Radio- (NH4)2SO4 to 80% saturation over 30min at 0°C chemical Centre, Amersham, Bucks., U.K.). The with constant stirring. The suspension was centri- reaction was terminated by the addition of 0.05ml fuged at 10000g for 20min in the MSE High Speed 18 of 2M-NaOH and the samples were reincubated for centrifuge and the pellet was dissolved in and dia- at least 1 h at 37°C. Portions (100,ul) were spotted on lysed against column buffer [containing (final con- to Whatman 3MM paper disks (2.5cm diam.), and centrations) 0.15M-KCI, 20mM-Tris-HCl buffer, these were washed six times in 5 % (w/v) trichloro- pH7.5, and 5mM-2-mercaptoethanol]. When the acetic acid in 50mM-Na4P207 and then dried with sample was to be applied to a DEAE-cellulose column ethanol and ether. The DNA was dissolved by it was further dialysed against 20mM-Tris - HCI heating with 0.5ml of 1 M-Hyamine hydroxide buffer, pH7.5, containing 5mM-2-mercaptoethanol. [Nuclear Enterprises (G.B.) Ltd., Edinburgh, U.K.] Table 1 gives details of the purification of the nuclear for 20min at 60°C in a counting vial. Radioactivity enzyme. was determined after the addition of toluene-based scintillator [0.5 % (w/v) 2,5-diphenyloxazole]. Column chromatography When diphosphates were used in the assay they replaced the triphosphates in equimolar amounts. Sephadex G-200 and Sepharose 4B were obtained When present, ATP was at 1.5mM. from Pharmacia (G.B.) Ltd., London W5 5SS, U.K. A unit of DNA polymerase activity is defined as Column chromatography was carried out at the amount required to catalyse incorporation of 4°C with a column (60cmxO.9cm diam.) that had 1 nmol of [Me-3H]dTTP into acid-insoluble material been calibrated with molecular-weight markers of in th at 37°C. cytochrome c, bovine serum albumin, haemoglobin, human y-globulin and urease. The flow rate was 5ml/h and 1 ml fractions were collected. DEAE-cellulose chromatography was carried out Nucleoside diphosphokinase assay in a column (6.8cm x 1.5cm diam.) with Whatman To assay the interconversion of di- and tri-phos- DE52 DEAE-cellulose (H. Reeve Angel and Co. phates 100,ul portions of the polymerase incubation Ltd., London EC4V 6AY, U.K.). The sample was mixtures were pipetted into 0.4ml of 5% trichloro- applied in and washed with 20mM-Tris-HCl buffer, acetic acid, and 50jul portions of the supernatant pH7.5, containing 5mM-2-mercaptoethanol, and then were chromatographed on Whatman no. 1 paper a 50ml linear gradient of 0-0.4M-KCI in the same with isobutyric acid-aq. NH3 soln. (sp.gr. 0.88)- buffer was applied. The column was run at 6.5ml/h EDTA (0.1M)-water (100:4.2:1.6:55.8, by vol.) and 1.3ml fractions were collected. (Krebs & Hems, 1952). Spots corresponding to 1973 NUCLEAR AND SOLUBLE DNA POLYMERASES 239 dTMP, dTDP and dTTP were cut out and radio- 0.5,umol of 2-mercaptoethanol and 20,tmol of either activity was counted by using toluene-based scintil- Tris-HCl buffer, pH7.5, or glycine-NaOH buffer, lator. pH9.2, in a total volume of 0.3ml. The reaction was terminated by cooling the samples to 0°C and 0.4mg Preparation of 14C-labelled DNA from L929 cells ofunlabelled salmon testis DNA was added as carrier. Acid-insoluble radioactivity was assayed with the aid L929 cells were grown in medium EC10 containing of toluene-based scintillator. Protein was determined [2-14C]thymidine [5OnCi (0.86nmol)/ml] for 3 days by the method of Lowry et al. (1951). Phospho- before they were harvested. The washed cells were lipase C and trypsin were obtained from Sigma suspended in balanced salt solution and poured into Chemical Co., St. Louis, Mo., U.S.A. Salmon testis an equal volume of 2% (w/v) sodium dodecyl and calf thymus DNA were obtained from sulphate containing 4mM-EDTA and 6% (w/v) of Worthington Biochemical Corp., Freehold, N.J., sodium p-aminosalicylate. The sample was stirred at U.S.A. 70°C for 30min and cooled to room temperature, and an equal volume of phenol-m-cresol (22:3, v/v) containing 0.1 % (w/v) 8-hydroxyquinoline was Results added.