THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 11, Issue ofJune 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,~ PETER BEARD,§ AND PAUL BERG From the Department of Biochemistry, Stanford Ilniuersity Medical Center, Stanford, California 94305 In uiuo-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(1), 5% to nicked, circular molecules (SV40(II)), 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. SV40 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. SV40 and polyoma DNA synthesis are currently the best understood examples of DNA synthesis in mammalian nuclei. SV40 DNA can be isolated from infected cells in five major forms; covalently closed superhelical circles (SV40(1), 80%; * This work was supported in part by Research Grants GM-13235 from the National Institutes of Health and ACSZVCZBD from the American Cancer Society. M. L. D. held National 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. 5 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(II) DNA, SV40 double-stranded circular DNA containing an interruption of the phosphodiester backbone in at least one of the two strands; SV4O(L) DNA, SV40 double-stranded linear DNA; SV4O(RI) 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 1 M NaCl leaving viral DNA in the supernatant following centrifugation. DNh-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(II)), 10%; circular replicating intermediates (SV40(RI)), 6%; linear molecules (SV4O(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 repIication 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 4341 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 hoth uninfected and virus-infected mammalian cells (27-34). The purpose of this paper is to describe an in uitro system that converts SV4O(RI) to the covalently closed, superhelical form SV40(I). An accompanying paper described how this system was used to detect and assay a cellular factor (or factors) required to convert SV40(RI) to SV40(1). 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 plates (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 pg/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 CHCIa per 20 parts virus suspension for 10 min at 4'. The top layer was removed after centrifugation for 3 min at 3000 x g, adjusted to 10% calf serum, and frozen at -20" in 2-mI aliquots. Virus stocks were titered by plaque assay using CV-1 cells. 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 pCi/ml, of 'Ti (carrier free) in phosphate-free medium containing calf serum dialyzed against 0.15 M NaCl or 10 pCi/ml of ['Hlthymidine (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 CHC1,- isoamyl 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 rnin at 0" in a Reckman 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 pg/ml) for 3 hours at 30" to digest RNA. SV40(I) and SV40(II) DNA were then isolated after neutral sucrose gradient sedimentation. With 'T 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(I) DNA isolated in this manner was indistinguishable from SV40(I) DNA that had been purified further by equilibrium sedimentation in a CsC1-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-HC1, pH 7.6, 1 mM Na,HPO,, 5 mM KCl, 137 mM NaCl, 0.5 mM MgCI,, and 0.9 mM CaCI,) 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 ['Hlthymidine (> 15 Ci/mmol) prior to preparation of cell lysates. When in vitro DNA synthesis was to be monitored by incorporation of [a-T]dATP or dCTP, then the SV40(1) DNA pool was prelabeled by adding 50 pCi of ['Hlthymidine to 5 ml of medium 2 hours before the cell lysates were prepared. Alternatively, replicating forms already present in vim, SV40(RI) DNA, were labeled by removing the medium, washing the 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 ['Hlthymidine per ml 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 M sucrose to prevent premature cell lysis and 0.1 mM thymidine to dilute residual ['Hlthymidine. Hypotonic buffer was 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.8, 5 mM KCI, 0.5 mM MgCI,, 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-ml centrifuge tube 0.15 ml of ce!l lysate was mixed with 0.05 ml 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 MgCt,, 0.5 mM dithiothreitol, 4 mM ATP, 5 mM phosphoenolpyruvate, 29 pg of pyruvate kinase, and 0.2 mM each of dATP, dGTP, dC'I'P, and d'I'TP to the final concentra- tions. In addition, the hypotonic buffer contributes 15 mM Hepes, pH 7.8, 3.7 mM KCI, 0.37 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 2.5 M 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 Coualently Ciosed 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 ('TIDNA containing about 50% SV40(I) and 50% SV(O(I1) DNA was added to the SDS superna- tant of the samples to he analyzed to serve as an internal standard. An aliquot of the SDS supernatant was sedirnented in an alkaline sucrose gradient as described below and two fractions collected; one containing SV40(I) 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 pg of salmon sperm DNA by adding 10 ml of cold 1 N HCI containing 0.510 sodium pyrophosphate and collected on Whatman GF/C glass fiber filters, washed three times with 10-ml portions of HCl-pyrophosphate solution followed by 5 ml of ethanol, then dried, and counted in a toluene scintillator. The percentage of SV40(I) ['HIDNA was corrected on the basis of the recovery of the SV40(I) ['T]DNA added as the internal standard. SI Nuclease Assay-SV40 ["PIDNA was added to the SDS super- natant fraction to serve as an internal standard and two 0.2-mI aliquots removed. The amount of acid-precipitable DNA was measured in one aliquot, and to the other was added: sonicated salmon sperm DNA (6 pl of 5 mg/ml), SDS (8 pl of 10% solution), and 0.186 ml of water to give final concentrations of 75 pg of DKNml, 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 (T, 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 ml 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 NaC1. 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(I) 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%. Sedimentdim Techniques Routine analysis of the amount of SV40(I) DNA made in uitro 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 CsCI-ethidium bro- mide density gradients, the SDS supernatants were extracted twice with 2 volumes of CHCI,-isoamyl alcohol (24:l) to remove protein. DNA samples (0.1 mlj 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 1 M NaCI, 1 mM EDTA, and 10 mM 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 %a+ 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 discs, dried, and then washed in batches thrce times in cold 1 M HC1,0.5% 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-HC1, pH 7.8, 1 mhi EDTA, CsCl of final density 1.565 dcc, and 400 pg 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 glcc 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-T]dA?'P, [a-'TIdCTP, and [a-'T]dGTP at specific activities of 50 to 80 Ciipmol, 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 PEINV 254) by ascending chromatography with fresh 0.4 M NH,HCO,. RESULTS Our preliminary studies on SV40 DNA synthesis in isolated nuclei provided two guidelines for this work. First, synthesis of SV40(I) 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(III)) prelabeled in uiuo into SV40(I) DNA in uitro. The following is a description of this system and an assessment of whether the conversion of SV40(RIi to SV40(I) DNA follows the same pattern of replication as occurs in uiuo. 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 9f% 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 [ 3H ]thymidine. 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 SV4O(Ij DNA compared tu 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 maximurn 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 SV4O(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, MgCI, was adjusted to 1 mM excess over the tot.al added nucleotides. An excess over 1 mM free Mg2+ was inhibitory with MgCl, concentrations above 7 mM. KCI 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,)$O, suspension, addition of more than 50 fig of enzyme TABLE I Requirements for SV40 DNA synthesis in uitro The standard assay contained 45 mM Hepes, pH 7.8, 44 mM KCI, 6.4 RIM MgCI,, 0.2 M sucrose, 0.9 mM dithiothreitol, 4 mM ATP, 5 mM phosphoenolpyruvate, 100 gg/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 [PH]thymidine in SV40(1) DNA went from 8 to 55% in 1 hour at 30". This conversion was defined as 100% activity. Conditions Complete standard assay .... ............ - ATP ................................... - dATP, dGTP, dCTP, dTTP ................... - Phosphoenolpyruvate and pyruvate kinase ......... - Pyruvate kinase .................... - Phosphoenolpyruvate ..................... - Sucrose ................................ - KC1 .......................................... + EDTA (2 mM) .................................. + EDTA(5mM) ..................................... + CaCI, (0.5 mM) ................................... + Spermine (5 mM) ................... + Spermidine (5 mM) ..................... + N-Ethylmaleimide (5 mM) .................. + UTP, GTP, CTP (50~~ each) + p-Chloromercuribenzoic acld (5 mMj ............. ................ Activity "a 100 60 30 55 80 72 70 60 67 5 80 19 23 <1