THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 11, Issue of June 10, pp. 4340-4347, 1975 Printed in U.S.A. Synthesis of Superhelical Simian Virus 40 Deoxyribonucleic Acid in Cell Lysates* (Received for publication, October 15, 1974) MELVIN L, DePAMPHILIS,t PETER Bearp,§ AND Paut BERG From the Department of Biochemistry, Stanford University Medical Center, Stanford, California 94305 In vivo-labeled SV40 replicating DNA molecules can be converted into covalently closed superhelical SV40 DNA (SV40(I)) using a lysate of SV40-infected monkey cells containing intact nuclei. Replication in vitro occurred at one-third the in vivo rate for 30 min at 30°. After 1 hour of incubation, about 54% of the replicating molecules had been converted to SV40(I), 5% to nicked, circular molecules (SV40(ID), 5% to covalently closed dimers; the remainder failed to complete replication although 75% of the prelabeled daughter strands had been elongated to one-genome length. Density labeling in vitro showed that all replicating molecules had participated during DNA synthesis in vitro. Velocity and equilibrium sedimentation analysis of pulse-chased and labeled DNA using radioactive and density labels suggested that SV40 DNA synthesis in vitro was a continuation of normal ongoing DNA synthesis. Initiation of new rounds of SV40 DNA replication was not detectable. $V40 and polyoma are well suited as simple models and biological probes of the mechanism of DNA replication in mammalian cells. Following infection by either virus, host enzymes involved in cellular DNA replication are induced (1). This induction of host cell DNA synthesis (2, 3) as well as the initiation of viral DNA replication (4, 5) requires a viral gene function. Elongation and termination of viral DNA replication appear to be entirely dependent on cellular gene products. Therefore, by studying SV40 DNA replication one might learn more about the details of cellular DNA replication. $V40 and polyoma DNA synthesis are currently the best understood examples of DNA synthesis in mammalian nuclei. SV40 DNA can he isolated from infected cells in five major forms; covalently closed superhelical circles (SV40(1),1 80%; *This work was supported in part by Research Grants GM-13235 from the National Institutes of Health and ACSZVC23D from the American Cancer Society. M. L. D. held Nationa! Science Foundation and National Institutes of Health postdoctoral fellowships during the course of this work. $ Present address, Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02115. § Present address, Institut Suisse de Recherches, Experimentales sur le Cancer, 1011 Lausanne (Suisse), Switzerland. ‘Abbreviations used are: SV40(I) DNA, SV40 double-stranded covalently closed, circular, superhelical DNA; SV40(I1) DNA, SV40 double-stranded circular DNA containing an interruption of the phosphodiester backbone in at least one of the two strands; SV40(L) DNA, SV40 double-stranded linear DNA; SV40(RJ) DNA, SV40 DNA replicating intermediate; BrdUTP, bromodeoxyuridine triphosphate; SDS, sodium dodecyl sulfate; SDS supernatant and SDS pellet, DNA isolated by the method of Hirt (6) which precipitates cellular DNA in 0.6% SDS and 1M NaCl leaving viral DNA in the supernatant following centrifugation. DNA-DNA hybridization studies showed that only 4% of the SV40(I) DNA is trapped in the pellet (P. Rigby, personal communication). Mitochondrial DNA also appears in the SDS super- natant but is labeled 0.1 to 1% as well as cellular or viral DNA (7, 46); Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid. relaxed circles containing a nick or gap in either strand (SV40(1}), 10%; circular replicating intermediates (SV40(RI)}, 6%; linear molecules (SV40(L}), 3%; and covalently closed dimers (1%). SV40(RI) DNA contains a superhelical region of unreplicated parental DNA, two relaxed loops participating in DNA synthesis (9-11) and the parental strands remain cova- lently closed (12). Newly synthesized strands are not cova- lently attached to parental DNA and are never longer than one-genome length (9-10). Replication forks may contain regions of single-stranded DNA (10, 13). To separate template strands during replication, a repeated nicking and resealing of parental DNA must occur, perhaps using the ‘‘relaxing factor” reported in uninfected mouse nuclei (14, 39). Replication of SV40 viral DNA begins at a unique point and proceeds bidirectionally at approximately equal rates (15-18). Chain elongation occurs discontinuously, apparently on both strands, through synthesis of short pieces 200 to 300 nucleo- tides long which are then joined into longer strands (19-22). In polyoma, viral DNA replicates semiconservatively (23) and the most newly synthesized DNA appears to contain RNA cova- lently attached to the 5’ terminus (12, 24). The mechanism for separation of circular progeny molecules at the end of replication is unknown, but clearly one of the template strands must be broken and rejoined. The only apparent intermediate during segregation of SV40 DNA is a circular DNA containing a nick in the daughter strand close to the normal termination site (25). Errors during replication as well as recombination are a likely explanation of dimeric forms (26). Since in vitro systems for SV40 DNA synthesis would allow greater control over biochemical parameters, purification, and characterization of DNA replication factors, and, most impor- tantly, complementation of cell and virus mutants in vitro, we 4340 have undertaken a study of SV40 DNA replication in nuclei from infected cells. Several cell-free systems have been de- scribed for the study of DNA replication in both uninfected and virus-infected mammalian cells (27-34). The purpose of this paper is to describe an in vitro system that converts SV40(RI) to the covalently closed, superhelical form SV40(1). An accompanying paper described how this system was used to detect and assay a cellular factor (or factors) required te convert SV40(RI) to SV40(). EXPERIMENTAL PROCEDURES Cell Lines CV1 cells obtained from S. Kit, MA-134 cells from J. Pagano, and BSC-1 cells from Flow Labs are established lines of African green monkey kidney cells. All were grown on plastic pilates (Nunclon or Falcon) in Dulbecco-modified Eagle’s Medium (Gibco) supplemented with 10% calf serum (Microbiological Associates), 500 units/ml of penicillin G, and 100 ug/ml of streptomycin sulfate in a CO; incubator at 37°, Virus Stock A plaque-purified isolate of the small plaque SV40 strain, Rh911 (35), was used in all experiments. Virus was grown on MA-134 cells by infecting at a multiplicity of 0.01 plaque-forming units per cell and harvesting the virus 10 to 12 days later as described by Estes et al. (36). Virus extracted from a polyethylene glycol precipitate was sterilized by shaking with 1 part CHCI, per 20 parts virus suspension for 10 min at 4°. The top layer was removed after centrifugation for 3 min at 3000 x &, adjusted to 10% calf serum, and frozen at —20° in 2-m) aliquots. Virus stocks were titered by plaque assay using CV-1 ceils. Preparation of Viral DNA Viral DNA markers were prepared from infected BSC-1 or CV-1 cells (multiplicity of infection of 40) labeled 24 hours post-infection with either 50 yCi/ml, of **P, (carrier free) in phosphate-free medium containing calf serum dialyzed against 0.15 M NaCl or 10 pCi/ml of (H]thymidine (20 Ci/mM) in normal medium. After 48 hours of infection, viral DNA was extracted by the method of Hirt (6). The sodium dodecyl! sulfate supernatants were extracted twice with CHCI,- isoamy! alcohol (24:1), and the DNA was precipitated with 5 volumes of ethanol at -20° overnight. The precipitate was collected by sedimentation at 23,000 rpm for 30 min at 0° in a Beckman SW 25.1 rotor, resuspended in 10 mM Tris, pH 7.8, 1 mM EDTA, and 0.1 m NaCl, then treated with pancreatic RNase (20 g/ml) for 3 hours at 30° to digest RNA. SV40(I) and SV40(II) DNA were then isolated after neutral sucrose gradient sedimentation. With "7P labeling, about equal amounts of SV40(I) and SV40(II) were recovered, whereas with *H labeling, 80 to 90% of the labeled DNA in the SDS supernatant was in SV40(1). SV40(1) DNA isolated in this manner was indistinguishable from SV40(1) DNA that had been purified further by equilibrium sedimentation in a CsCl-ethidium bromide density gradient. Growth and Infection of Cells for in Vitro DNA Synthesis BSC-1 cells were generally used both because they gave the most active preparations and cell DNA synthesis is not stimulated by SV40 infection (37). Cells were seeded in 9-cm plastic dishes at a density of 5 x 10° cells per dish. The medium was changed 2 or 3 days later and the cells were infected for 1 hour on Day 4 or 5 at a multiplicity of 40 plaque-forming units of stock virus per cell in 0.6 ml of TS buffer (20 mM Tris-HCl, pH 7.6, 1 mm Na,HPO,, 5 mM KCl, 137 mm NaCl, 0.5 mM MgCl,, and 0.9 mm CaCl,) containing 5% calf serum. Fresh medium was added and incubation at 37° continued for 35 hours at which time cell lysates were prepared for in vitro DNA synthesis. Standard Conditions for Preparation of Cell Lysates Infected cells were labeled routinely with [*H]thymidine (>15 Ci/mmol) prior to preparation of cell lysates. When in vitro DNA synthesis was to be monitored by incorporation of [a-"7P]dATP or dCTP, then the SV40(I) DNA pool was prelabeled by adding 50 »Ci of [*H thymidine to 5 ml of medium 2 hours before the cell lysates were prepared. Alternatively, replicating forms already present in vivo, SV40(RI) DNA, were labeled by removing the medium, washing the 4341 cell monolayers with 10 ml of TD buffer (TS buffer without MgCl, and CaCl,), and addition of 0.5 ml of TD buffer containing 100 pCi of ("H]thymidine per m! and flotation of the dishes on a 37° water bath for 3.5 min. At this time 0.5 ml of TD buffer containing 4 mm EDTA and 1% trypsin was added. The plates were incubated an additional minute at 37° then floated on ice water to arrest further DNA synthesis. All subsequent steps were carried out between 2 and 4°, using ice baths whenever possible. One milliliter of TD buffer was added to each dish and the cells were removed with the aid of a pipette, pooled, adjusted to 20% calf serum to inhibit the trypsin, then sedimented at 2000 x g for 3 min. The pellet was resuspended in 4 ml of hypotonic buffer per dish of cells supplemented with 0.2 mM sucrose to prevent premature cell lysis and 0.1 mM thymidine to dilute residual [*H]thymidine. Hypotonic buffer was 20 mM N-2-hydroxyethy!piperazine-N’-2-ethanesulfonic acid, pH 7.8, 5 mm KCl, 0.5 mm MgCl,, and 0.5 mM dithiothreitol. The cells were sedimented again and then resuspended in hypotonic buffer at about 2 x 10’ cells per ml. The cells were allowed to swell for 8 min at 0° then lysed by four strokes of a tight fitting Dounce homogenizer (Kontes Glass Co., pestle B) and the extract used without further fractionation. Standard Conditions for in Vitro DNA Synthesis In a 5-m] centrifuge tube 0.15 ml of cell lysate was mixed with 0.05 m] of an assay mix at 2°. The assay mix contributed 0.20 M sucrose, 30 mM Hepes, pH 7.8, 40 mm KCl, 6 mm MgC}, 0.5 mM dithiothreitol, 4 mM ATP, 5 mm phosphoenolpyruvate, 29 ug of pyruvate kinase, and 0.2 mM each of dATP, dGTP, dCTP, and dTTP to the final concentra- tions. In addition, the hypotonic buffer contributes 15 mm Hepes, pH 7.8, 3.7 mm KCl, 6.37 mm MgCl, and 0.37 mM dithiothreitol, Where indicated labeled deoxynucleoside triphosphates were present at 0.02 mM. The reaction was terminated after incubation for 1 hour at 30° by addition of 0.4 ml of 20 mM Tris, pH 7.8, 40 mm EDTA, and 1.2% SDS. When the preparation was completely solubilized, 0.4 ml of 2.6 mM NaCl was added and the tubes stored at 4° for at least 8 hours to precipitate cellular DNA (6) which was then removed by sedimentation at 17,000 x g for 40 min. DNA found in the SDS supernatant was greater than 90% viral judged by sedimentation analysis of infected and uninfected cell lysates. This agrees with other reports where DNA-DNA hybridiza- tion assays were also performed (38). Rapid Assays for Covalently Closed DNA Two methods were routinely used to determine the fraction of SV40(I) DNA present in the SDS supernatants: sedimentation in alkaline sucrose gradients (9) and S1 nuclease digestion following heat denaturation of the DNA (40). Alkaline Sucrose Sedimentation—SV40 [*?P JDNA containing about 50% SV40(1) and 50% SV40(I) DNA was added to the SDS superna- tant of the samples to be analyzed to serve as an internal standard. An aliquot of the SDS supernatant was sedimented in an alkaline sucrose gradient as described below and two fractions collected; one containing SV40() DNA and the second containing SV40(II + L) DNA, nascent host DNA, and viral DNA released from replicating molecules. The labeled DNA in each fraction was precipitated with 25 wg of salmon sperm DNA by adding 10 ml of cold 1 N HCl containing 0.5% sodium pyrophosphate and collected on Whatman GF/C glass fiber filters, washed three times with 10-ml portions of HC]-pyrophosphate solution followed by 5 ml of ethanol, then dried, and counted in a toluene scintillator. The percentage of SV40(I) [7H]DNA was corrected on the basis of the recovery of the SV40(I) [*7P ]DNA added as the internal standard. S1 Nuclease Assay—SV40 [**P JDNA was added to the SDS super- natant fraction to serve as an internal standard and two 0.2-ml aliquots removed. The amount of acid-precipitable DNA was measured in one aliquot, and to the other was added: sonicated salmon sperm DNA (6 xt] of 5 mg/ml), SDS (8 ul of 10% solution), and 0.186 m] of water to give final concentrations of 75 ug of DNA/ml, 0.2% SDS, and to dilute the NaCl present to 0.5 M. Samples were heated at 100° for 6 min to denature nicked forms of SV40 DNA (Tm of DNA containing 40% G + C is 95° in 0.5 M NaCl). Following rapid cooling in ice water, sodium acetate (0.2 ml! of 0.6 M, pH 4.6), zinc acetate (0.14 ml of 0.05 m, pH 4.6), and 1.26 m! of water were added to give final concentrations of 60 mM sodium acetate, pH 4.6, 3.5 mM zinc acetate, and 0.1 M NaCl. Enough single strand-specific S1 nuclease (prepared by the method of Sutton (41)) was added to digest the single-stranded DNA in 5 to 10 4342 min at 37°, although the incubation was generally for 25 min before precipitating the resistant DNA. Calculations of the fraction of SV40(1) DNA were normalized with respect to the recovery of the internal standard of ["P]DNA. Results using either method were in excellent agreement. The average deviation of triplicates containing 50% SV40(I}) DNA was +1.5%. Sedimentation Techniques Routine analysis of the amount of $V40(I) DNA made in vitro was done directly on SDS supernatants as described above. To characterize the sedimentation behavior of newly synthesized DNA, the samples were dialyzed for 12 hours against two changes of 10 volumes of 10 mM Tris, pH 7.8, 1 mm EDTA, and 0.1 M NaCl before layering over sucrose gradients. Prior to equilibrium centrifugation in CsCl-ethidium bro- mide density gradients, the SDS supernatants were extracted twice with 2 volumes of CHC1,-isoamy} alcohol (24:1) to remove protein. DNA samples (0.1 ml) were layered on linear 5 to 20% sucrose gradients in 4.2 ml of polyallomer tubes and sedimented at 4° in a Beckman SW 56 rotor at 55,000 rpm for the indicated times. Neutral gradients contained 1M NaCl, 1 mm EDTA, and 10 mo Tris, pH 7.4. Alkaline gradients contained 5 mm EDTA, 0.2 to 0.8 M NaOH (proportional to sucrose concentration), 0.8 to 0.2 M NaCl (to make the Na? concentration up to 1 M). Fractions of 3 to 10 drops were collected from the bottom through a 20-gauge needle onto 2.5-cm diameter Whatman No. 3MM paper dises, dried, and then washed in batches three times in cold 1 MHC], 0.5% sodium pyrophosphate (5 to 10 ml per disc), and then twice in ethanol. After drying, the discs were counted in a toluene scintillator. Equilibrium density gradient centrifugation was performed either in a solution containing 10 mM Tris-HCl, pH 7.8, 1 mM EDTA, CsCl of final density 1.565 g/cc, and 400 yg of ethidium bromide per ml to separate SV40(I) DNA from other forms of DNA or in the same solution except with a final density of 1.700 g/cc and no ethidium bromide. Gradients (6 ml total volume) were formed during centrifugation in a Beckman 50 Ti rotor at 37,000 rpm for 55 hours at 4°. Fractions were collected from the bottom either in test tubes or on Whatman No. 3MM discs and washed as described above. Reagents {a-*7P JGATP, {a-*7P]dCTP, and [a-**P]dGTP at specific activities of 50 to 80 Ci/umol, were prepared by the method of Symons (42, 43). 5-Bromodeoxyuridine triphosphate was synthesized by the method of Chamberlin and Berg (44) and kindly donated by Klaus Geider. All other reagents were obtained from commercial sources. Chromatography Labeled nucleotides were separated on polyethyleneimine thin layer strips (Brinkmann Polygram CEL 300 PEI/UV 254) by ascending chromatography with fresh 0.4 m NH,HCQs. RESULTS Our preliminary studies on SV40 DNA synthesis in isolated nuclei provided two guidelines for this work. First, synthesis of SV40(]) DNA was more efficient in crude cell lysates than in purified nuclei, and second, that incorporation of radioactive deoxynucleoside triphosphates into DNA found in SDS super- natants was not a reliable indicator of SV40 DNA replication (45). Therefore, a crude cell lysate system was developed and evaluated entirely on its ability to convert replicating SV40 DNA (SV40(RI)) prelabeled in vive into SV40(I) DNA tn vitro. The following is a description of this system and an assessment of whether the conversion of SV40(RI) to SV40(I) DNA follows the same pattern of replication as occurs in vivo. Conditions for DNA Synthesis in Cell Lysates—The rate of viral DNA synthesis in BSC-1 cells was about twice that observed in CV-1 or MA-134 cells and reached a maximum at 32 to 44 hours after infection. At that time greater than 95% of the cells were positive for T antigen (47). BSC-1 cells have the further advantages that SV40 infection does not induce cell DNA synthesis and the cells can be removed rapidly from the culture dishes by trypsinization following a 3.5-min pulse of (°H Jthymidine. Three methods for lysing cells were compared. Cells were removed routinely by trypsinization, swelled in a hypotonic buffer, and then lysed in a Dounce homogenizer as described under “Experimental Procedures.” In this way large numbers of cells could be collected and lysed just prior to use. An alternative method avoided trypsin by scraping osmotically swollen cells from their plates with a rubber policeman and then dispersing aggregated nuclei in a loose fitting Dounce homogenizer. This method gave equivalent results but was more time-consuming when large numbers of dishes were involved. A third approach involved trypsinization and lysis in 0.2% Triton X-100, Brij-58, or Nonidet P-40 detergents. These lysates produced about 50% less SV40(1) DNA compared to mechanically prepared lysates because some SV40({(RI) was converted to SV40(II) DNA; however, endogenous SV40(I) DNA present before detergent was added was unaffected. Cell lysates prepared 32 to 42 hours after infection gave the maximum conversion of SV40(RI) to SV40(1) RNA, and were stable for at least 3 hours at 2°. At earlier times, e.g. 20 hours, conversion was only 70% as efficient. Table I shows the conditions needed for optimal conversion of SV40(RI) to SV40(I) DNA. Because of contributions from the cell lysate, omission of any one component did not have a drastic effect on the reaction. The ATP concentration was optimal from 2 to 8 mm. At each ATP concentration, MgCl, was adjusted to 1 mM excess over the total added nucleotides. An excess over 1 mM free Mg?* was inhibitory with MgCl, concentrations above 7 mm. KCl had a broad optimum between 30 and 80 mm (Fig. 1). The sulfate and ammonium ions were inhibitory even though the pH remained constant during the assay. Since pyruvate kinase was used as an (NH,).SO, suspension, addition of more than 50 yg of enzyme Tas_e | Requirements for SV40 DNA synthesis in vitro The standard assay contained 45 mm Hepes, pH 7.8, 44 mm KCI, 6.4 mM MgCl,, 0.2 m sucrose, 0.9 mM dithiothreitol, 4 mm ATP, 5 mm phosphoenolpyruvate, 100 ug/ml of pyruvate kinase, and 0.2 mM each of dATP, dGTP, dCTP, and dTTP. Activity refers to the conversion of prelabeled SV40(RI) to SV40(I) DNA measured as described under “Experimental Procedures.” In the standard assay the percentage of (‘H]thymidine in SV40(I) DNA went from 8 to 55% in 1 hour at 30°. This conversion was defined as 100% activity. Conditions Activity % Complete standard assay .......... 000.0... 00.0 100 — ATP oon cece eee 60 — dATP, dGTP, dCTP, dTTP .......0...0 2.2. 30 ~ Phosphoeno)lpyruvate and pyruvate kinase .......... 55 — Pyruvate kinase ....... 2000000000000... 80 ~ Phosphoenolpyruvate .........0.000.0.00...00.... 72 — Sucrose 2.0.0... cence 70 OO 5 O) 60 + EDTA (2mM) 2200. 67 + EDTA (5mM) 0.00. cee § + Cal, (0.5 mM) 20.2 eee 80 + Spermine (5 mM) .... . bccn eects 19 + Spermidine (6mM) ...0000.000 0000000 eee 23 + N-Ethylmaleimide (5 mM) .......0...............-. 5 1 1 1 1 § : 700 200 Salt Concentration, mM Fic. 1. Effect of salt concentration on conversion of SV40(RI) to SV40(1) DNA. Using the standard assay described under “Experimen- tal Procedures,” the amount of KCl present was varied. K,SO, was then substituted for KCl. (NH,);SO, was added to the standard assay containing 44 mM KCl. The percentage of [*H]thymidine in SV40(I) DNA before incubation in vitro was 9%. per ml also inhibited DNA synthesis. Even with added ATP, the presence of an ATP regenerating system composed of phosphoenolpyruvate and pyruvate kinase stimulated forma- tion of SV40(I) DNA. Increasing the deoxyribonucleotide concentrations above 200 um did not increase the rate of SV40(1) DNA synthesis. In a typical experiment the amount of ['H]thymidine in SV40(I) DNA varied from 7 to 10% of the total acid-insoluble label at the start of incubation and reached a maximum of 50 to 60% by 1 hour at 30° (Fig. 2). The initial rate of SV40(I) DNA synthesis in vitro was approximately 60% of that ob- served in vivo during a pulse chase at the same temperature. Based on initial rates of synthesis in vivo, about 50% of the SV40(RI) was converted to SV40(I) DNA in 16 min; one round of replication in vivo required 30 min at 30°. The period of DNA synthesis in vitro was not increased by adding more of the assay components after 40 min of incuba- tion at 30° although sufficient nucleotides were still present at this time to incorporate [a-*7P]dGTP into added DNase- treated salmon sperm DNA. The fate of deoxynucleotides in the complete assay system was examined directly using [a- “’PJdGTP. After 60 min at 30°, 20% of the remaining unincor- porated label chromatographed as dGMP, 35% as dGDP, and 45% as dGTP. In contrast, when 2 mm ATP was present with- out a regenerating system the initial rate of dGTP disappear- ance was 3-fold faster and resulted in 80% dGMP, 15% dGDP, and 5% dGTP after 1-hour incubation. This suggests that one function of ATP and an ATP regenerating system was to maintain the nucleotide pools. Efforts to improve the synthetic capacity of a cell lysate by increasing enzyme stability included addition of bovine serum albumin, an acidic protein (1 to 12 mg/ml), cytochrome c, a basic protein (1 to 12 mg/ml), and polymers such as Ficoll (10 mg/ml), polyethylene glycol 6000 (10 mg/ml), dextran 500 (10 mg/ml), and glycerol (0.1 to 1.0 M) in place of sucrose. These agents either had no effect or caused a 10 to 20% inhibition at the highest concentrations tested. Small amounts of ionic polymers such as dextran sulfate (0.5 mg/ml) and DEAE-dex- tran (0.5 mg/ml) resulted in 75% inhibition. Some nuclear enzymes may have been lost or diluted in the preparation of lysates. However, a cell lysate could be diluted with the assay mix as much as 40-fold with no decrease in the amount of SV40(I) DNA synthesis. In addition, supplementa- tion of the lysates with Escherichia coli DNA polymerase I (10 4343 & 8 3 oe S % sv4o CD DNA o 8 ng 5 = o $ J 30. 40~~=50 60 Minutes Fic. 2. Time course for conversion of SV40(RI) to SV40(1) DNA at 30°. Infected cells were pulse-labeled for 4 min with ([*H ]thymidine as described under “Experimental Procedures.” Lysates were prepared and assayed in the standard way. The remaining cell monolayers were washed twice with cold TS buffer containing 500 um thymidine then covered with 10 ml of TS buffer containing 5% calf serum and 100 pM thymidine. The dishes were floated on a 30° water bath. Reactions were stopped by adding 1 ml of 0.6% SDS, 20 mm EDTA, and 10 mm Tris (pH 7.8) to a 100-cm dish of cells, and the SDS supernatant prepared and analyzed for the fraction of (H]thymidine in SV40(I) DNA. The same conversion of SV40(RI) to SV40(1) DNA was obtained with cell monolayers covered with 10 ml of Dulbecco’s Modified Eagle’s Medium plus 10% calf serum and incubated at 37° for 1 hour with 6% CO;. units (48)) alone or together with E. coli DNA ligase (2 units (49}) and NAD (30 uM) did not increase the amount of SV40(1} DNA made. Although heparin and E. coli DNA polymerase I stimulated DNA synthesis in isolated rat liver nuclei 10- to 20-fold (50), we found that heparin (100 ug/ml) alone inhibited formation of SV40(I) DNA by 90%, while addition of both heparin and DNA polymerase I showed no difference from the control lysate. RNA synthesis may be required to initiate discontinuous elongation of viral DNA (24). However, addition of ribonucleo- side triphosphates (Table I) did not stimulate production of SV40(I) DNA. Rifampicin (20 to 200 ug/ml), a specific inhibi- tor of E. coli RNA polymerase (51), showed no inhibition of either incorporation of [a-**P JdATP into viral DNA or conver- sion of SV40(RI) to SV40(I) DNA. A rifampicin derivative, AF/ABDP (20 yg/ml), reported to inhibit gene amplification in Xenopus (51-53) and in vitro synthesis of ¢X174 parental RF,? also had no effect. About 80% of the inhibition observed at higher drug concentrations could be accounted for by the residual dimethylsulfoxide used to dissolve the inhibitors. Since proteolytic enzymes in the lysate could prevent further in vitro DNA synthesis, both toluene sulfonyl fluoride (0.01 to 1 mM), a general inhibitor of serine proteases, and tosyl-1- phenylalanyl chloromethy] ketone (0.01 to 0.1 mM), a specific inhibitor of chymotrypsin, were added but these had no effect on either viral DNA synthesis or formation of SV40(I) DNA in vitro. Characterization of Viral DNA Replication Products Made in Vitro—SV40(RI) DNA was labeled with [*H ]thymidine in intact cells in order to analyze its conversion into SV40(1) DNA in a cell lysate. Following a 4.5-min pulse of [5H ]thymidine, only 8% of the *H label found in the SDS supernatant was in 7W. Wicker, personal communication. 4344 SV40(I) DNA; the remainder sedimented between 18S and 4S in an alkaline sucrose gradient (Fig. 3A). About 85% of the viral [SHJDNA sedimented at 26 S in a neutral sucrose gradient, the position characteristic of SV40(RI) DNA (9), while about 5% was SV40(II) DNA (Fig. 4A). Moreover, about 80% of the viral SH{DNA] banded at a buoyant density expected of SV40(RI) DNA in a CsCl-ethidium bromide gradient (Fig. 5A), i.e. intermediate between that of SV40(1) and SV40(II) DNA (9). When uninfected cells were labeled and processed like infected cells, only 2% as much labeled DNA appeared in the SDS supernatant and it was generally found distributed in the bottom third of the neutral sucrose gradients as previously reported (9). After incubation of the pulse-labeled infected cell lysate for 1 hour at 30° under standard conditions, the amount of *H label in SV40(I) DNA increased to 55% as judged by the amount of 3H Cem eo Sage ne. 0 Fi. 3 (upper left). Alkaline sucrose gradient sedimentation of SV40 DNA synthesized in vitro. BSC-1 cells infected with SV40 were incubated for 4 min at 37° with [*H thymidine to predominantly label SV40(RI) DNA. Cell lysates were prepared and incubated in vitro for 9 or 60 min as described under ‘Experimental Procedures.” Alkaline sucrose gradients were overlaid with 0.1 ml of the SDS supernatant fraction dialyzed against 10 mM Tris (pH 7.8), 1 mM EDTA, and 0.1 M NaCl. A **P-labeled SV40 DNA standard containing both SV40(I) and SV40(II) DNA was then added to the sample and the gradient centrifuged in a Beckman SW 56 rotor at 55,000 rpm, 4°, for 2 hours. Fractions were collected from the bottom of the gradient. O-——, °H; @-—e, *’P: J, SV40(1) DNA; ZZ, SV40(II) DNA; covalently closed dimers appear in Fractions 6 to 8 after 50 min of incubation in vitro. Fic. 4 (upper center). Neutral sucrose gradient sedimentation of $V40 DNA synthesized in vitro. Procedure was that described in Fig. 3 except gradients were centrifuged for 3.25 hours. O——O, °‘H; @-——@, *P; [, SV40(1) DNA; HZ, SV40(ID DNA. Fic. 5 (upper right), CsCl-ethidium bromide density equilibrium gradient of SV40 DNA synthesized in vitro. Procedure was that ae i! OMIN 1 t be} i i lus ; aa ; : - ™ z a comin i Fo gq 6 6 4 42 2 e658 10 20 wo a Fraction A 7 = linears. OMIN 0 SS circles ) ‘ 6 a ie mi OS 6 iif * ‘ ‘i | ‘ 9 2 2 : 7 6 60 MIN 7 3 % $s 6 No 6 a 0 y 10 20 30 Fraction 40 Fraction described in Fig. 3 except that the sample was centrifuged to equilibrium in CsCl-containing ethidium bromide as described under “Experimental Procedures.” O——-O, ‘H; @——®, °*P; f, SV40(D DNA; JZ, SV40(ID) DNA. Fic. 6 (lower left). Neutral sucrose gradient sedimentation of SV40 DNA removed from the denser band of Fig. 5. SV40(I) PHJDNA taken from a 60-min pulse-chase experiment such as described in Fig. 3 was isolated from the lower band in CsCl-ethidium bromide gradient such as described in Fig. 5 and then sedimented through a neutral sucrose gradient. A **P-labeled standard SV40 DNA sample containing both SV40(I) and SV40(II) DNA was added to the sample after it was layered on the gradient. O——O, 7H; @ @, *P; J, SV40(1) DNA; I, SV40(II) DNA. Dimeric DNA is found in Fractions 4 to 7. Fic. 7 (lower right). Separation of single-stranded circular and sin- gle-stranded linear viral DNA. The same procedure described in Fig. 3 was followed except that an alkaline sucrose gradient was centrifuged for 6.5 hours. A °*P-labeled SV4Q(II) DNA standard was added to the sample after it was layered on the gradient. O——-O, *H; @——®, *’P. SV40(]) DNA observed in either alkaline gradient sedimenta- tion (Fig. 3B) or CsCl-ethidium bromide equilibrium centrifu- gation (Fig. 5B); more than 98% of the SV40(I} DNA isolated from density equilibrium gradients also behaved as SV40(I) DNA on neutral (Fig. 6) and alkaline sucrose gradients (data not shown). The smail discrepancy occurred during handling the DNA since an SV40(I) [*7PJDNA internal standard was damaged to the same extent. Thus, SV40(1) DNA produced in vitro was indistinguishable from SV40({I) DNA synthesized in vivo. Following this 1-hour incubation in vitro, about 55% of the 5H label was identified as SV40(I) DNA, 10% as SV40(II), 5% as covalently closed dimers, and the remaining 30% as SV40(RI). SV40(1I) DNA was identified on neutral sedimenta- tion gradients (Fig. 4B), while covalently closed dimeric DNA was detected in alkaline sedimentation gradients (Fig. 3B), and neutral sedimentation gradients of covalently closed DNA taken from CsCl-ethidium bromide gradients (Fig. 6}. The remaining *H-labeled viral DNA was very likely SV40(RI) DNA because it sedimented faster than the SV40(I) peak in neutral sucrose gradients (Fig. 4B) and banded at an interme- diate density in CsCl-ethidium bromide gradients (Fig. 5B). These may be molecules which were initiated in vivo but failed to complete replication in vitro. Some of the apparently unfinished SV40(RD DNA may also be nicked circular dimeric DNA which would sediment at about 21.5 S at pH 7 in 1 mM NaCl and have a buoyant density in CsCl-ethidium bromide gradi- ents identical with SV40(ID DNA. Although the data presented in Figs. 3 to 6 show the nature and extent of the change in viral DNA after 1 hour of incubation, the same analysis with samples taken earlier during the incubation confirmed the progressive conversion of prelabeled SV40(RI) to SV40(I) DNA expected from Fig. 2. With time, pre-existing daughter strands are elongated; at the beginning about 67% of the SV40(RI) [SHJDNA was in DNA strands shorter than one SV40 length (Fig. 7A), but after a 1-hour incubation at 30°, 75% of the *H label was in strands of S$V40 length (Fig. 7B). Incorporation of Labeled Nucleotides into SV40 DNA—The incorporation of [a-*7P]JdATP and [a-**P dCTP was used to characterize intermediates in the conversion of SV40(RI) to S$V40(1) DNA in vitro. Lysates from infected cells, prelabeled for 1 hour with [*H]thymidine, were prepared and incubated with the a-**P-labeled deoxynucleoside triphosphates. About 90% of the *H label appeared in SV40(I) DNA which was neither degraded nor nicked during in vitro incubation with P-labeled substrates since the ratio of SV40(I) to SV40(II) DNA remained unchanged (Fig. 8). During the incubation the *2P label was incorporated about equally into the SDS superna- tant and the SDS pellet. Following a 10-min incubation, about 90% of the *P label in the SDS supernatant was in SV40(RI) DNA (Fig. 8A), and about 5% was in SV40(I) DNA as judged by the S1 nuclease assay. Fifty minutes later, 35% of the **P in the SDS supernatant was in SV40(J) DNA and about 5% sedimented as covalently closed dimers (Fig. 9). The remaining **P label was in SV40(RI) DNA which had only been partially completed (Fig. 8B). Occasionally some of the **P label (<25%) was found to sediment between 3 S and 7S (Fig. 8), but there was never a peak of *H label from the prelabeled SV40(RI) [(*H JDNA in this region of a neutral sucrose gradient. The significance of this is discussed later. The conversion of SV40(RI) to SV40(1) DNA in vitro was 30 4345 10 MIN N 32p cpm x 102 3H CPMx 10°3 8 Fraction Fic. 8. Neutral sucrose gradient sedimentation of SV40 DNA labeled during in vitro synthesis. Cell lysates were prepared from BSC-1 cells infected with SV40 and incubated 1 hour at 37° with (*H ]thymidine to label predominantly SV40(I1) DNA and then incu- bated in vitro for 10 or 60 min in the presence of [a-*7P dATP and [e-°*P|dCTP. The SDS supernatant fraction was dialyzed, 0.1 ml layered over a neutral sucrose gradient, and centrifuged in a Beckman SW 56 rotor, 4°, 55,000 rpm for 3.25 hours. O_O, *H; @——®, ™P; J, $V40() DNA; H, SV40(11) DNA. to 40% less efficient when measured with *7P-nucleotides than with *H-prelabeled SV40(RI1). This was expected because those SV40(RI) molecules which had only just begun replication would incorporate the greatest amount of [a-*?P]dATP and those closest to completion of replication would contain the greatest amount of *H label and also have the highest probability of becoming SV40(I) DNA. Note that 70% of the "P-labeled single strand, linear viral DNA that had not entered SV40(I) by 1 hour had elongated to complete SV40 DNA length (Fig. 10). Results determined by both labeling techniques were consistent with the conclusion that most growing DNA chains proceed to completion in vitro as they would have in vivo and about 50% of the nearly completed SV40(RI) continue onto SV40(1) DNA. DNA Synthesis in Presence of BrdUTP—The previous experiments indicate that SV40 DNA molecules that had already begun replication in vivo continue to be replicated in vitro. A similar conclusion was obtained by density labeling. Cell lysates were prepared from SV40-infected cells that had been labeled in two ways. The first was labeled in vivo with (‘H]thymidine for 1 hour at 37° and the second for 4 min. In the first, 90% of the *H label was in SV40(1) DNA whereas in the second 85% of the *H label was in SV40(RI) DNA. These lysates were prepared and incubated for 30 min under the standard conditions (see “Experimental Procedures”) except that BrdUTP (10 mM) replaced dTTP in the reaction mixture and the temperature was 37°. As expected, the density of 4346 60 MIN a i SO ee