Vol. 16, No. 1 Printed in U.S.A.

JOURNAL OF VIROLOGY, JUlY 1975, p. 70-74 Copyright 0 1975 American Society for Microbiology

Fate of Polyoma Form I DNA During Replication ANN ROMAN1* AND RENATO DULBECCO2

Salk Institute for Biological Studies, San Diego, California 92112; and Department of Biology, University of California, San Diego, La Jolla, California 92037 Received for publication 26 December 1974

The fate of polyoma form I DNA generated during replication was investigated in resting BALB-3T3 cells. The experiments showed that there was extensive re-entry of such molecules into replication. This process took place over a period of several hours and appeared to be random. Progeny form I molecules must, therefore, spend some time in a nonreplicating pool before reinitiating replication. We propose that two factors affect the fate of progeny form I DNA. (i) The rate of reinitiation of progeny molecules is determined by the capacity of the initiation machinery. (ii) The extent of re-entry is determined by the availability of maturation proteins which divert form I from replication. the Department of Biology, University of California, San Diego.)

The experiments of Hirt (5) on the semiconservative replication of polyoma DNA and the observations of Bourgaux et al. (3) that there is a lag before labeled cyclic strands are seen in the replicative intermediate (RI) of polyoma suggested that daughter molecules of the RI, referred to as progeny form I, reinitiate replication. Progeny form I DNA may participate in replication in one of the following ways. (i) All DNA molecules are in a continuous state of replication; i.e., a progeny form I molecule as soon as completed immediately initiates a new round of replication. (ii) Some progeny form I molecules are diverted to a nonreplicating pool where they spend some time before re-entering the replicating pool. If form I molecules derived from RIs immediately re-enter replication, infected cells would contain very little or no form I. Bourgaux et al. (3) and Roman et al. (9) have shown that after a relatively long labeling time or after a chase form I molecules accumulate. Such results show that a nonreplicating pool exists. Experiments in this report were designed to follow the fate of progeny molecules after they have entered the nonreplicating pool. We present evidence that form I molecules re-enter replication over an extensive period of time. The implications of such kinetics on the sequence of events in virus replication are discussed. (The data reported in this paper represent a portion of a Ph.D thesis by A. R. submitted to

MATERIALS AND METHODS Cells. BALB-3T3 cells (1) (1.5 x 106 per 9-cm NUNC petri dish) were plated in 10 ml of Dulbecco modified eagle medium (DME) containing 10% calf serum. At 5 to 6 days after seeding the cultures were exposed to polyoma virus (100 PFU/cell) for 1 h. The cultures were then covered with DME lacking serum. Viral DNA replication began 16 h after infection, reaching a maximum at 34 h after infection. Virus. Virus stocks of the large plaque strain of polyoma (10) were made through infection of primary baby mouse kidney cells (12) at an input multiplicity of 0.5 to 1 PFU/cell. Cells and medium were collected on day 6 after infection and subjected to low-speed centrifugation. The supernatant was used in the experiments to be described. Labeling and extraction of DNA. At 24 h postinfection cells were labeled for 20 min with 50 to 100 uCi of [3H ]thymidine per ml (specific activity 20 Ci/ mmol). At the end of this time cells were rinsed twice with DME and covered with medium containing unlabeled 10-6 M thymidine in DME. After 30 min the plates were again washed twice with DME, and the cells were covered with DME containing 5 x 10-5 M BUdR, 2 x 10-" M 5-fluorodeoxyuridine (11), and 9 x 10-8 M deoxycytidine (2, 8). At varying times viral DNA was extracted by the method of Hirt (6). The medium was removed, and 0.8 ml of a solution containing 0.01 M Tris, 0.01 M EDTA, 0.6% sodium dodecyl sulfate, pH 7.2, was added. After 10 min at room temperature, 0.2 ml of 5 M NaCl was added to the plate and mixed into the lysate by gentle swirling. ' Present address: Department of Microbiology and Immu- The lysate was poured into centrifuge tubes and at 4 C overnight. Subsequently, each tube nology, University of Oregon Medical School, Portland, Ore. stored was centrifuged for 30 min at 15,000 rpm in the 97201. 2Present address: Imperial Cancer Research Fund Labora- Sorvall centrifuge. The supernatant is referred to as tories, POB 123, Lincoln's Inn Fields, London WC2A 3PK, the Hirt supernatant. Analysis of viral DNA. Form I DNA was sepaEngland. 70


VOL. 16, 1975


aliquot of each Hirt supernatant was counted. The results (Fig. 1) indicate that BUdR decreases the rate of DNA synthesis only slightly; viral DNA synthesis in the presence of BUdR was 70% that of synthesis in the presence of thymidine. Kinetics of re-entry of progeny form I into replication. Twenty-four hours postinfection viral DNA was labeled as described in Materials and Methods. DNA from some cultures was extracted at the end of the cold thymidine chase RESULTS. to determine the amount of label present in Effectiveness of the cold thymidine chase. replicating molecules. DNA from the remaining To determine the length of chase necessary to cultures was extracted at various times after the complete molecules in the process of replication addition of the BUdR mixture to determine the at the end of the radioactive pulse, cells were proportion of 3H-labeled DNA which re-entered labeled for 20 min with [3H ]thymidine at 24 h replication. Aliquots of the Hirt supernatant after infection, and the label was chased for were layered on 5 to 20% alkaline sucrose varying lengths of time with 10- 6 M cold gradients. Polyoma form I DNA is denatured thymidine. Viral DNA was extracted, and the irreversibly under these conditions. Fractions total acid-precipitable counts in the Hirt super- from the form I peak were pooled, neutralized, natant were measured at various times over the and banded to equilibrium in CsCl. next hour. The proportion of label in form I was determined by alkaline sucrose gradient sedimentation. There was only a moderate increase 0 400 in incorporated counts during the chase (Table 1). The proportion of label in form I increased during the first 20 min of chase and then 350kremained constant. Therefore, a chase period of rated from form II and RI DNAs by layering samples onto 5 to 20% alkaline sucrose gradients containing 0.25 M NaOH, 0.75 M NaCl, and 10-3 M EDTA. The samples were centrifuged in the SW41 rotor at 32,000 rpm for 4 h. To separate HL DNA (hybrid DNA with bromodeoxyuridine [BUdR] substitution in one strand) from LL DNA (light DNA with no BUdR substitution) the peak fractions from the alkaline sucrose gradients were pooled and neutralized. CsCl was added to give a final density of 1.72 g/ml. The samples were spun in the type 40 rotor at 33,000 rpm for 48 h.

30 min was selected. Effect of BUdR on the rate of DNA synthesis. In the experiments to be described BUdR was used as a density label to follow the fate of molecules labeled with [3H]thymidine. To verify that in our experiments DNA synthesis was unaffected by incubation with BUdR, cells were covered with either 5 x 10-5 M BUdR, 2 x 10-5 M 5-fluorodeoxyuridine, 9 x 10- 6 M deoxycytidine or 5 x 10-5 M thymidine, 2 x 10-5 M 5-fluorodeoxyuridine, 9 x 10- 6M deoxycytidine at 24 h postinfection. Incorporation in the first mixture was followed by [3H ]BUdR and in the second by [3H ]thymidine. At various times after addition, viral DNA was extracted and an



x U

200 IL (J




TABLE 1. Incorporation of [SH]thymidinea during a chase with 10-6 M thymidine Length of 10-1 M thymidine chase (min)

Hirt supenatant


% Form I

36 5,374 55 5,787 77 5,900 73 6,003 77 7,186 76 7,898 a Infected cells were first pulsed for 20 min with 50 1sCi of [8H ]thymidine per ml. 0 10 20 30 45 60














FIG. 1. The effect of BUdR on the rate of DNA synthesis. At 24 h after infection mixture (i) 5 x 10-6 M BUdR, 2 x 10-s M 5-fluorodeoxyuridine, 9 x 10- 6 M deoxycytidine or (ii) 5 x 10-I M thymidine, 2 x 10- M 5-fluorodeoxyuridine, 9 10-6 M deoxycytidine was added to BALB-3T3 cultures. At the indicated times viral DNA was extracted and an aliquot of the Hirt supernatant was counted. DNA synthesis in mixture a (0) was monitored with [8H]BUdR and in mixture b (0) with [3H]thymidine. x



Analysis of the CsCl centrifugation (Fig. 2) showed [3H ]thymidine associated with two peaks: LL unsubstituted DNA and HL hybrid density DNA. The following data suggest that the more dense material is truly HL DNA and not produced by breakdown of 'H-labeled DNA and re-utilization of the radioisotope. (i) The density difference between the HL and LL peaks is approximately 0.045 g/ml, suggestive of material with full BUdR substitution in one strand (5). (ii) There is very little material between the two peaks; it would seem fortuitous to find all breakdown products in the HL density position. (iii) No HH DNA molecules were seen when the form I molecules were banded at higher CsCl concentration, which would have retained them within the gradient. These should be the predominant species if there were breakdown and re-utilization. To determine the kinetics of re-entry of form I into replication the percentage of HL DNA (HL/HL + LL) was calculated and is shown in Fig. 3. At the end of the 10- 6 M thymidine chase in this experiment, 89% of the ['H ]thymidine was associated with form I DNA. To determine what proportion of HL molecules arose from the amount of label found in replicating molecules before addition of BUdR, HL/RI was calculated. It is difficult to obtain an accurate value for RI at the end of the chase. An approximation has been obtained by determining the number of counts sedimenting more slowly than form I. Certainly this is a maximum value, since most of the counts in the form II region probably derive from breakdown of form I (9). HL is equal to [(percentage of HL at the end of the BUdR chase) x (the number of counts in form I at the end of the cold thymidine chase)]. Since HL/RI is always greater than one, the number of counts in HL form I DNA must not have been derived solely from the counts in RI DNA at the end of the cold thymidine chase. Similar experiments done at different times after infection yielded results generally similar to those of Fig. 3. DISCUSSION In the experimental procedure label was permitted to enter viral DNA for a brief time. After incubation in 10-6 M thymidine to allow labeled replicating molecules to complete, BUdR was added to density-label newly synthesized DNA. This protocol leads to daughter form I molecules which contain a 3H-labeled parental strand and a BUdR-labeled newly synthesized daughter strand. Therefore, the HL 3H-labeled molecules extracted at a given time correspond to those originally labeled molecules that have undergone at least one new round of replication


during the BUdR chase. LL 'H-labeled molecules are those which have not undergone replication during the same period. As the number of counts in HL form I DNA at the end of the BUdR chase is greater than RI at the end of the 10-6 M thymidine chase (Fig. 3), those counts could only be the result of re-entry of form I molecules into replication. Two main characteristics of the re-entry process are evident: (i) re-entry is extensive, since it affects about half of the pulse labeled DNA; and (ii) molecules generated during a short time interval (20 min) return to the replicating pool over a period of several hours, suggesting that the phenomenon is random. The leveling off of the curves after several hours in BUdR deserves special comment. It could derive from accidental events connected with the experimental procedure. This includes (i) cell damage by the biochemically potent substances used or by starvation of cells or (ii) decreased efficiency of BUdR incorporation with time. The evidence (Fig. 1) for continued incorporation of BUdR at a constant rate during the period when re-entry of form I into replication appears to cease suggests neither of these is the major contributor to che observed cessation. Further evidence that altered BUdR incorporation is not a major factor is shown in Fig. 2.. After 8 h in the presence of BUdR there are two discrete peaks of radioactivity, the density difference between the two peaks is the same as earlier in the chase, and there is very little material between the two peaks. If the efficiency of BUdR incorporation was decreasing with time one would expect to see a shift in density of 'H-labeled, BUdR-substituted DNA toward the lighter unsubstituted peak (4, 5). Alternatively, the cessation of re-entry could reflect characteristics of the infectious process. To discuss the re-entry curves as representative of the infectious process we make the assumption, supported by the kinetic data in Fig. 3, that labeled and unlabeled molecules are indistinguishable from one another. Thus, the proportion of labeled molecules that undergo replication within a certain time is equal to the proportion of all molecules that replicate within that time. A decreased rate of re-entry of labeled molecules occurs when one or both of the following conditions apply: (i) the pool of form I expands so that the machinery necessary for re-entry into replication is saturated and the re-entry of all such molecules slows down; (ii) molecules of form I are irreversibly blocked from re-entering replication by becoming committed to maturation. Expansion of the pool leads to cessation of re-entry of the labeled mol-

350 300

250 200-

100 50


250 -B

CL~~ 200




100 50-


ioo00 50D


a-150100 50E




25 35 45 55 65 FRACTION NUMBER FIG. 2. CsCl density equilibrium gradient centrifugation of radioactive hybrid (HL) form I DNA and light (LL) form I DNA. Twenty-four hours after infection, BALB-3T3 cells were labeled for 20 min with [3HJthymidine, chased for 30 min with 10-6 M thymidine, and further chased for varying lengths of time with 5 x 10-' M BUdR, 2 x 10- M 5-fluorodeoxyuridine, 9 x 10- ' M deoxycytidine. Viral DNA was layered onto 5 to 20% alkaline sucrose gradients. Fractions from the form I peak were pooled and neutralized, and CsCI was added to adjust the density to 1.72 g/ml. Samples were centrifuged in the type 40 rotor at 33,000 rpm for 48 h. Fractions were precipitated with 5% trichloroacetic acid, and their radioactivity was determined. CsCl density increases from right to left. The peak of lower density corresponds to unlabeled, irreversibly denatured form I DNA. The difference in density between the two peaks, as measured by refractive indexes, is 0.045 g/ml. The length of BUdR chase was as follows: (A) 30 min; (B) 1 h; (C) 2 h; (D) 4 h; (E) 8 h. 73




I 60 0 0

00 -0,~~~~~~~





20[ -Q..t




FIG. 3. Relationship between length of BUdR chase and the conversion of LL form I DNA to HL form I DNA. The percentage of HL was calculated from Fig. 2 (1-, 3-, and 10-h BUdR chase not shown). The ratio of HL/RI is equal to [(% HL at the end of the BUdR chase) x (the number of counts in form I at the end of the cold thymidine chase) Vthe number of coutnts in non-form I material at the end of the cold thymidine chase.

ecules only when the number of DNA molecules greatly exceeds the capacity of the replicating machinery. Figure 1 shows that the incorporation of label into DNA over the time course of this experiment increases by at most a factor of 2.7 by 28 h postinfection, at which time re-entry appears to have ceased. Therefore, it is unlikely that pool expansion could be responsible for the cessation. If, however, labeled molecules were irreversibly removed from the nonreplicating pool, the re-entry of form I molecules as measured by radioactivity would cease when all labeled molecules are withdrawn from the pool. As the amount of viral DNA which is encapsidated over the period of time of this experiment is less than 10% of the total labeled form I (A. Roman, unpublished data), there must be accumulation of DNA which has been blocked from re-entering replication but is not totally encapsidated. We propose that the availability of both initiation protein(s) and maturation protein(s) determines the fate of progeny form I DNA. This is reflected in the kinetics of re-entry. The efficiency of the intiation machinery determines the initial slope of the re-entry curve; the maturation machinery is reflected in the maximum extent of re-entry, percentage of HL. It is likely that the rate of formation of RI occurs initially at an accelerated rate in individual cells if the replicating pool is to expand. Later in infection the constant rate of accumulation of form I DNA suggests that a constant number of molecules undergoes replication. At this time


form I molecules may be involved not only in replication but also in partial encapsidation. This model would predict that the slope of the re-entry curve and the extent of re-entry would be greatest when the experiment is initiated early in infection. Our preliminary data support this prediction. If the experiment is initiated 20 h postinfection, when viral DNA is accumulating at an accelerated rate, the initial slope of the re-entry curve is steeper and the final extent of re-entry greater than at 24 or 28 h postinfection, when the rate of DNA synthesis is constant. The suggestion of a limited reinitiation capacity is consistent with the report of Manteuil et al. (7), who concluded that the number of replication sites in growing CV-1 cells infected with simian virus 40 is limited and that the probability of replicated molecules to re-enter replication decreases with time after infection. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant CA 07592 from the National Cancer Institute. LITERATURE CITED 1. Aaronson, S. A., and D. J. Todaro. 1968. Development of 3T3-like lines from Balb/c mouse embryo cultures: transformation susceptibility to SV40. J. Cell. Physiol. 72:141-148. 2. Ben Porat, T., C. Coto, and A. S. Kaplan. 1966. Unstable DNA synthesized by polyoma virus-infected cells. Virology 30:74-81. 3. Bourgaux, P., D. Bourgaux-Ramoisy, and R. Dulbecco. 1969. The replication of ring-shaped DNA of polyoma virus. I. Identification of the replicative intermediate. Proc. Natl. Acad. Sci. U. S.A. 64:701-708. 4. Calothy, G., K. Hirai, and V. Defendi. 1973. 5Bromodeoxyuridine incorporation into simian virus 40 deoxyribonucleic acid. Effects on simian virus 40 replication in monkey cells. Virology 55:329-338. 5. Hirt, B. 1966. Evidence for semiconservative replication of circular polyoma DNA. Proc. Natl. Acad. Sci. U.S.A. 55:997-1004. 6. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell culture. J. Mol. Biol. 26:365-369. 7. Manteuil, S., J. Pages, D. Stehelin, and M. Girard. 1973. Replication of simian virus 40 deoxyribonucleic acid: analysis of the one-step growth cycle. J. Virol. 11:98-106. 8. Reichard, P., Z. N. Canellakis, and E. S. Canellakis. 1961. Studies on a possible regulatory mechanism for the biosynthesis of deoxyribonucleic acid. J. Biol. Chem. 336:2514-2519. 9. Roman, A., J. J. Champoux, and R. Dulbecco. 1974. Characterization of the replicative intermediate of polyoma virus. Virology 57:147-160. 10. Vogt, M., and R. Dulbecco. 1962. Studies on cells rendered neoplastic by polyoma virus: the problem of the presence of virus-related materials. Virology 16:41-51. 11. Weil, R., M. R. Michel, and G. K. Ruschman. 1965. Induction of cellular DNA synthesis by polyoma virus. Proc. Natl. Acad. Sci. U.S.A. 53:1468-1475. 12. Winocour, E. 1963. Purification of polyoma virus. Virology 19:158-168.

Fate of polyoma form IDNA during replication.

Vol. 16, No. 1 Printed in U.S.A. JOURNAL OF VIROLOGY, JUlY 1975, p. 70-74 Copyright 0 1975 American Society for Microbiology Fate of Polyoma Form I...
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