JOURNAL OF VIROLOGY, Feb. 1975, p. 259-267 Copyright @ 1975 American Society for Microbiology

Vol. 15, No. 2 Printed in U.S.A.

Replication of Polyoma DNA in Isolated Nuclei V. Complementation of In Vitro DNA Replication BERND

OTTO

AND

PETER REICHARD*

Medical Nobel Institute, Department of Biochemistry, Karolinska Institute, S-104 01 Stockholm,

Sweden

Received for publication 4 October 1974

Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA. When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands. When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations, DNA synthesis was decreased. In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments. All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract. Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E. coli DNA polymerase I) increased the joining function of the nuclei. The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication. We described earlier an in vitro system for the study of polyoma DNA replication, consisting of isolated nuclei from 3T6 mouse fibroblasts infected with polyoma virus (15). During incubation, the nuclei are able to continue and complete the elongation of progeny strands which were initiated in vivo (9). This process was shown to proceed in a stepwise fashion and to involve the intermediate formation of Okazaki type fragments (11) about 150 nucleotides long, which are initiated at their 5' end by short stretches of ribonucleotides (2, 8). Experiments by Mueller and co-workers demonstrated that the in vitro synthesis of chromosomal DNA in nuclei from HeLa cells could be stimulated by cytoplasmic factors (4, 10). Here we explore the possibility of influencing the synthesis of a viral DNA in isolated cell nuclei by addition of cytoplasmic extracts or purified enzymes. With this system it is possible to demonstrate effects on discrete steps participating in the overall process, and our experiments should provide a complementation system similar to what has been described for microorganisms (7).

versity of Tiibingen, Tiubingen, Germany, 1970) was a gift from Heinz Schaller. Calf thymus DNA ligase I, purified 1,000-fold by the method of Soderhall and Lindahl (13) and then further purified by chromatography on phosphocellulose, was a gift from Stefan Soderhall. Most of the methodology including the handling of cells and virus has been described previously (15). In vivo labeling of DNA. Immediately before the preparation of nuclei, cells were incubated for 6 to 7 min with [3H]TdR (1.0 MM) in the culture medium. Labeling was terminated by removal of the medium and addition of 5 ml of ice-cold Tris-buffered saline (15). Under these conditions, about 90% of the radioactivity was recovered in the progeny strands of

MATERIALS AND METHODS [3H ]thymidine ( [3H ]TdR; 6.7 Ci/mmol) and [a32PJdGTP were obtained from New England Nuclear Corp.; ["4C]dATP was purchased from Amersham. Escherichia coli DNA polymerase I (fraction VII. Jovin et al. [6]) was a gift from Lambert Skoog; E. coli polynucleotide ligase was prepared essentially by the method of Olivera and Lehman (12) with some minor modifications (B. Heyden, Dipl. Arbeit, Uni259

replicative intermediates. Preparation and incubation of nuclei. The preparation of nuclei from five petri dishes (15 cm), each containing approximately 107 cells, is described. All manipulations were performed at 4 C. (i) Normal nuclei: the Tris-buffered saline was removed, and the cell monolayers were washed two times with 5 ml of isotonic N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid (HEPES) (15). Cells were scraped and diluted into one 15-ml centrifuge tube with 5 ml of isotonic HEPES, and Nonidet P-40 was added to a final concentration of 0.5%. The suspension was vortexed at 2-min intervals, first for 90 s and then five times for 30 s on a Lab Line Super Mixer. Isotonic HEPES (10 ml) was added, and the suspension was centrifuged for 10 min at 800 x g. The pellet containing the nuclei (about 0.5 ml) was resuspended with 1.5 ml of isotonic HEPES and used either directly for incubation or stored at -70 C after quick freezing in an ethanol-dry ice-bath. (ii) Depleted nuclei: frozen suspensions of normal nuclei prepared from about 5 x 107 cells were thawed and diluted into 20 ml of

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isotonic HEPES. Nonidet P-40 was added to a final tures stimulated incorporation of radioactive concentration of 0.5%, and the suspension was vor- deoxynucleoside triphosphates into DNA only texed five times for 30 s as described above. After marginally and under special conditions (8). centrifugation for 10 min at 800 x g, the nuclear Probably the normal isolated nuclei retain conpellet was resuspended in 1.5 ml of isotonic HEPES. siderable pools of ribonucleoside triphosphates. The depleted nuclei were then frozen and stored at When ribonucleoside triphosphates other than -70 C. Incubation conditions. The conditions previously ATP were added to depleted nuclei, both the described (15) were modified as follows: [a-"32P]deox- rate and extent of DNA synthesis were stimuynucleoside triphosphate (4,000 to 8,000 counts per lated (Fig. 1). Incubation mixtures in all further min per pmole) or ["4C ]dATP (700 counts per min per experiments contained ribonucleoside triphospmole) were used at saturating concentrations (10 phates at saturating concentrations unless MM), and ribonucleoside triphosphates were always stated otherwise. included at a concentration of 60 MM (except for ATP, Dependence of DNA synthesis on concenwhich was present at 2 mM). Further variations of the tration of nuclei and stimulation by addition incubation conditions are given for individual experiments. All incubations were at 25 C. Selective extrac- of cytoplasmic extract. In the following experition of viral DNA according to Hirt (5) was performed ment, the concentration of nuclei (normal or depleted) in the incubation mixture was varied, as described previously. Preparations of cytoplasmic extracts. All manip- and the amount of DNA synthesis was deterulations were performed at 4 C. Between 26 and 28 h mined. As described above, the replicative inafter infection, the cell monolayers were washed twice termediates in nuclei were prelabeled with tritwith 5 ml of ice-cold Tris-buffered saline per dish and ium, while the in vitro incorporation utilized then covered with 5 ml of isotonic HEPES. After 5 [a- 32P]dGTP. The 32P to 3H ratio is thus a min, buffer was removed and plates were left in a vertical position for another 5 min to drain. The cells measure of in vitro DNA synthesis. This ratio was drastically decreased at low concentrations were scraped off and treated five times at 2-min intervals with three strokes in a loose-fitting Dounce of nuclei. The effect was apparent with both homogenizer. This highly concentrated lysate was types of nuclei, even though it was somewhat centrifuged for 40 min at 25,000 x g. NaCl was added more pronounced with the depleted nuclei (Fig. to the supernatant to give a final concentration of 2). Addition of higher concentrations of dithio0.1 M. The supernatant was dialyzed for 24 h against threitol (up to 10 mM) had no effect. The isotonic HEPES supplemented with 0.1 M NaCl and results suggest that at low concentrations of was then frozen at -20 C. About 0.5 to 1.0 ml of nuclei the activity of the system is limited by cytoplasmic extract was obtained from 5 x 107 cells. Determination of DNA synthesis. The in vivo some factors which are lost from the nuclei. The ['H]TdR prelabel described above was used to stan- effect was less pronounced during short incubadardize the amount of nuclei used in the in vitro tion times (5 min), indicating that factors were incubations. For each preparation of nuclei, the eluted from nuclei during DNA synthesis in specific activity (3H counts per min per Ag of DNA) was determined first. From this value and from the 1.0 amount of 'H label in a given experiment we could I then calculate the amount of nuclei used. This value is expressed as Hg of DNA per 100 Al of incubation mixture. The in vitro DNA synthesis is correlated to -.

the in vivo prelabel and expressed as the ratio of "2P or 4C label to 'H label.

Alkaline sucrose gradient centrifugation. These all run in a Beckman SW 56 rotor for 3 h at 55,000 rpm at 4 C. An internal marker (16S) of labeled, linear polyoma DNA obtained by cleavage of form I with E. coli R, restriction enzyme (1) was usually included. were

I

t

0.5

U)-

10

20

30

MINUTES

RESULTS

Stimulation of DNA synthesis of depleted nuclei by ribonucleoside triphosphates. Although RNA synthesis appears to be required for the elongation of polyoma DNA in isolated nuclei (8), the addition of ribonucleoside triphosphates other than ATP to incubation mix-

FIG. 1. Effect of ribonucleoside triphosphates on DNA synthesis of depleted nuclei. Depleted nuclei (27 ug of DNA) were incubated for different times with and without 60 MM each of CTP, GTP, and UTP. Standard conditions were modified as follows: deoxyribonucleoside triphosphates were used at 10 MM, ATP at 0.4 mM final concentrations. Symbols: 0, with 60 MM each of CTP, GTP and UTP; and *, without CTP, GTP, and UTP.

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The polyoma-specific nature of the DNA synthesized in depleted nuclei in the presence of extracts was demonstrated by reannealing experiments (Table 1). Equal relative amounts of in vitro 32P-labeled DNA were annealed to Ic ,' polyoma DNA independent of whether DNA was synthesized in the absence of extracts or in the presence of extracts (39%). Sim(42%) 20 60 s0 ilar results were obtained with the in vivo 3HCONCENTRATION OF NUCLEI (jig DNA/loo jA FIG. 2. Dependence of viral DNA synthesis on labeled DNA which hybridized to 37% and 32%, concentration of nuclei. The indicated amounts of respectively. In a parallel experiment, authentic nuclei (micrograms of DNA/100-microliter volumes) polyoma DNA reassociated to about the same were incubated under standard conditions for 30 min. extent (43%). Symbols: 0, depleted nuclei; and *, normal nuclei. Sedimentation properties of in vitro synthesized DNA. In the following sections we will vitro as well as during preparation of nuclei. show that the effect of extract also is of a This interpretation is also favoured by an exper- qualitative nature and in particular reflects the iment (data not shown) in which depleted ability of the nuclei to join small DNA pieces to nuclei were first incubated for 4 min under longer chains. In Fig. 4, two alkaline sucrose standard conditions but without labeled deox- gradient centrifugations of the products syntheynucleoside triphosphates. After pelleting, dif- sized by normal and depleted nuclei, respecferent amounts of nuclei were resuspended in tively, are compared. After a 30-min incubation standard incubation mixtures containing "2P- with normal nuclei, most of the incorporated in labeled dGTP and DNA synthesis was mea- vitro label was found in long chains sedimenting sured for 25 min. In such experiments, DNA close to 16S while with depleted nuclei about synthesis (32P to 3H) was low and showed less dependence on the concentration of nuclei. 1.5

,I

40

These results suggested that DNA synthesis of diluted nuclei could be restored by the addition of factors lost from them, and that such factors might be present in cytoplasmic extracts. This approach assumes that nuclei can exchange proteins with the incubation medium. The effect of cytoplasmic extracts on in vitro DNA synthesis is shown in Fig. 3. Two concentrations of nuclei were incubated for 30 min with increasing amounts of extract. In both cases DNA synthesis was stimulated, but the degree of stimulation was much larger with the lower concentration of nuceli. The extent of DNA synthesis obtained with the largest amount of extract was the same, however, and was identical to that observed with high concentrations of nuclei in the absence of extract (Fig. 2). The amount of extract required to obtain half maximal stimulation was about three times larger with the lower concentration of nuclei. Cytoplasmic extracts used in this and all further experiments described were prepared from polyoma-infected 3T6 cells, but similar effects were obtained with extracts from growing, uninfected cells. Extracts heated for 5 min at 100 C lost their stimulating capacity. Furthermore, the stimulating capacity could be fractionated by ammonium sulfate precipitation and precipitated between 30 and 60% saturation. Both of these results suggest that the stimulating factors are proteins.

5

10

15

20

CYTOPLASMIC EXTRACT (i's)

FIG. 3. Effect of cytoplasmic protein extract on DNA synthesis of depleted nuclei. Prelabeled, depleted nuclei were incubated at two concentrations of nuclei with increasing amounts of cytoplasmic protein extracts from polyoma-infected cells (microliters). Incubations were carried out under standard conditions for 30 min. In parallel incubations, DNA synthesis of indicated amounts of extracts per se was measured. DNA synthesis (32P/3H) of nuclei was corrected for the incorporation values of extract alone. The largest correction (20 uliters of extract) amounted to 15%. The protein concentration of the extract was about 10 mg/ml. Symbols: 0, 27 Ag of DNA/100 Aliters final volume; and *, 13 lAg of DNA/100 Mliters final volume.

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TABLE 1. Reannealing to polyoma DNA bound to filtersa Sample

Depleted nuclei Depleted nuclei + 10Mliters of extract Depleted nuclei + 15 uliters of extract Polyoma DNA

DNA synthesis Input radioactivity 3 32P/3H 32p

0.55 1.03 1.42

180 462 522

(counts/min)

Reannealing (% )b

14C

324 438 366 910

32P

3H

42 39 39

37 32 32

"C

43

The conditions for reannealing were the same as previously described (15). 'Filters contained 0.5 gg of polyoma DNA.

a

half of the radioactivity appeared as 4 to 5S fragments. These short fragments were earlier shown to be precursors of long strands (8). The results (Fig. 4) suggest that depleted nuclei are lacking in the capacity to join the fragments. A second point concerns the amount of 3H sedimenting in the position of form I DNA (53S). When the values for the fraction of prelabeled DNA sedimenting at 53S were corrected for the amount of prelabel in form I DNA by the in vivo pulse (12%), an additional 12% of the in vivo labeled viral DNA was transformed to form I DNA after a 30-min incubation. The corresponding value for depleted nuclei was only 2%. Addition of cytoplasmic extracts restored the capacity of depleted nuclei to join 4 to 5S fragments. Figure 5 shows the effect of two concentrations of extract on the sedimentation profile in alkaline sucrose gradients. In both cases a considerable stimulation of total DNA synthesis had occurred, and is expressed by an increased amount of in vitro label (321p) appearing as long single-stranded chains sedimenting close to 16S and as form I DNA (53S). In the absence of nuclei, the extract alone showed a limited capacity to synthesize DNA (Fig. 5B), most of which sedimented as short chains. This activity probably explains the shoulder at the 4 to 5S position in Fig. 5C and D. The addition of extract also considerably increased the amount of prelabel in form I DNA, which was stimulated from 2%T (Fig. 5A) to 11% (C) and 19% (D). Effects of some purified enzymes on DNA synthesis of depleted nuclei. It appeared to be of interest to investigate whether the addition of certain purified enzymes, either alone or in combination, could mimic the effect of crude cytoplasmic extracts. Depleted nuclei clearly are deficient in the ability to join short DNA chains. Since this deficiency might in turn be the cause of the general inhibition of strand elongation, we first investigated whether polynucleotide ligase might substitute for the cytoplasmic extract. Addition of highly purified ligase from either calf thymus or E. coli did not

increase the total amount of in vitro label

incorporated into polyoma DNA. However, there was a slight effect on the distribution of isotope in alkaline sucrose gradients involving a

shift of some of the material present as short DNA fragments to longer chains (Fig. 6B and C). Addition of more ligase did not increase these effects. However, addition of DNA polymerase I from E. coli together with ligase increased this shift (Fig. 7D and E). There was now also a slight stimulation of total amount of DNA synthesis (up to 125% of the control) but the amount of stimulation observed with cyto-

plasmic

extracts

(Fig. 5)

was

definitely not at-

tained. Similarly, there was only a slight stimulation of the amount of isotope appearing in form I. Our results demonstrate that the process of polyoma DNA synthesis in isolated nuclei can be influenced by purified enzymes and that depletion of ligase and DNA polymerase are involved in the loss of synthetic capacity of the nuclei. Since E. coli DNA polymerase I has both polymerizing and exonucleolytic activities (14) both might be involved in the observed stimulatory effect. However, even though these enzymes were added in large excess, they could by no means completely restore the activity of the nuclei, suggesting that cytoplasmic extracts supply other factors as well. The stimulatory effects described above suggest that purified enzymes and effectors present in cytoplasmic extracts can penetrate into isolated nuclei and participate in the replication process inside the nuclei. Alternatively, DNA and enzymes may diffuse out from the nuclei, and the stimulations observed in the presence of extracts may thus represent processes occurring outside the nuclei. To distinguish between these two alternatives the following experiment was done. Depleted nuclei were incubated either alone or in the presence of an excess of DNA ligase from calf thymus or cytoplasmic extract. At the end of the incubation, DNA synthesized outside the nuclei was separated from the DNA synthesized inside by centrifugation. About 10% of the total in vitro incorporated radioactivity

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COMPLEMENTATION OF IN VITRO DNA REPLICATION

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40 20 FRACTION NUMBER FROM BOTTOM FIG. 4. Characterization of viral DNA synthesized in normal and depleted nuclei. Prelabeled nuclei (36 ,g of DNA of normal nuclei and 27 ,g of DNA of depleted nuclei) were incubated under standard conditions with 14C-labeled dATP for 30 min. Portions (200 Mliters) of Hirt supernatant fluid were centrifuged through an alkaline sucrose gradient as described in text. Panel A, normal nuclei; panel B, depleted nuclei. Symbols: 0, 3H; and *, "C.

was found outside the nuclei when they were incubated alone or in the presence of ligase. The amount increased to 27% in the presence of extract. Figure 7 shows the sedimentation profiles in alkaline sucrose gradients of the DNA synthesized in the nuclei. It is clear that similar effects to those observed in Fig. 5 and 6 were found. A comparison of Fig. 7C and 5D suggests that the radioactive material present as a shoulder in the position of 4 to 5S DNA fragments in Fig. 5D was outside the nuclei. In conclusion, the addition of cytoplasmic

extract indeed stimulated the overall elongation process of DNA replication inside depleted nuclei. DISCUSSION The capacity of nuclei to complete the elongation of progeny strands during incubation depended on the concentration of nuclei. The relative amount of polyoma DNA synthesis was greatly decreased when low concentrations of nuclei were used. This effect was more pronounced with nuclei which had been prepared

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B

-

0

C 100

0

,* 0~~~~~~~~~ I~~~~~~ *0

0~~~~~~~~ 600~~~~~~~~

*16 S

20 40 0 20 FRACTION NUMBER FROM BOTTOM

40

FIG. 5. Effect of cytoplasmic protein extracts on DNA synthesis of depleted nuclei (product analysis). Prelabeled, depleted nuclei (13 ,ug of DNA) were incubated either in the absence (A) or presence of 20Mliters (C) or 50 Mliters (D) of cytoplasmic protein extract from polyoma-infected 3T6 cells. A 50-gliter amount of extract was also incubated without nuclei (B). All incubations were for 30 min under standard conditions with [32P]-labeled dGTP. The data were obtained from identical portions (200 ,liters of Hirt supematant fluid with 5 .liters of "'C-labeled linear polyoma DNA as a marker) run in parallel on alkaline sucrose gradients (see text). The arrows give the position of the marker (16S). Symbols: 0, 3H; and o, S2p.

I,

X I

20 20 400 FRkCTION NR FROM WTT

40

FIG. 6. Effect of some purified enzymes on DNA synthesis of depleted nuclei. Prelabeled, depleted nuclei (13 or calf thymus DNA ligase I (C); or with E. coli DNA polymerase I in the presence of either calf thymus DNA ligase I (D) or E. coli polynucleotide ligase (E). Incubations were for 30 min. The standard incubation mixture with [82P]-labeled dGTP was supplemented with NAD+ (10-I M final). Portions (200 /uliters) of Hirt supernatant fluid together with 5 /Lliters of "4C-labeled linear polyoma DNA as a marker were centrifuged through alkaline sucrose gradients (see text). The arrows give the position of the marker (16 S). Symbols: 0, 3H; and o, 32P. 265

gg of DNA) were incubated without added enzymes (A); with E. coli polynucleotide ligase (B)

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20 40 40 20 FRACTION NUMBER FROM BOTTOM FIG. 7. Effect of ligase or cytoplasmic protein extract on DNA synthesis inside depleted nuclei. Prelabeled depleted nuclei (13 ug of DNA) were incubated under standard conditions with [32P]-labeled dGTP for 30 min: (A) alone, (B) with DNA ligase I from calf thymus, or (C) with 50 ,liters of cytoplasmic protein extract from polyoma-infected mouse fibroblast 3T6 cells. DNA synthesis was stopped by adding 100 Mlliters of ice-cold isotonic HEPES to the incubation mixture. After centrifugation for 10 min at 4 C at 800 x g, the nuclear pellet was suspended in 100 gliters of isotonic HEPES and viral DNA from both the supernatant and the nuclei suspension was extracted by the Hirt procedure (5). Identical portions (200 Iliters of Hirt supernatant fluid from the nuclei fraction and 5 /.liters of '4C-labeled linear polyoma DNA as a marker) were run in parallel in alkaline sucrose gradients (see text). The arrows give the position of the marker (16S). Sym bols: 0, 3H; and , S2P.

by repeated washing with buffers containing Nonidet P-40 and which were therefore less contaminated with cytoplasm. The decrease was mainly apparent in the capacity of nuclei to sustain DNA synthesis and did not so much affect initial rates. From our data, it seems likely that part of the enzymes are washed out during preparation of nuclei and part leave the nuclei during DNA synthesis. Nevertheless, depleted nuclei incubated at a high concentration retained their capacity to continue the elongation process. Earlier results showed that polyoma DNA synthesis in nuclei initially involved the formation of short DNA fragments, 100 to 150 nucleotides long, and that these fragments subsequently were joined to form longer progeny strands (8). With depleted nuclei we now find an accumulation of short fragments also during

prolonged incubation indicating that some of the components being removed are involved in the joining process. The synthetic capacity can be fully restored by addition of cytoplasmic extracts, and the active factors in the extracts appear to be proteins. Restored DNA synthesis leads to the formation of long progeny strands. The extracts thus supply enzymes required for the joining of the short DNA fragments as well as for other steps of the elongation process. Extracts also increase the rate of transformation of replicative intermediate-DNA into mature viral DNA (form I DNA), but it is uncertain whether extracts also supply enzymes for the segregation of completely replicated DNA molecules. Formation of form I was, however, never as efficient as was reported for crude lysates of polyomainfected 3T3 cells (3).

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COMPLEMENTATION OF IN VITRO DNA REPLICATION

About equal activities were found with exor uninfected growing 3T6 cells. This result indicates that at least all enzymatic activities which can replace functions in the elongation process are of cellular rather than of viral origin. Polynucleotide ligase is an enzyme which would be expected to participate in the joining process. Neither purified calf thymus ligase nor E. coli ligase stimulated the overall capacity of the nuclear system to synthesize polyoma DNA. However, a shift of isotope from short fragments to long progeny strands was observed. This result indicates that ligase activity probably was one of the depleted nuclear factors, but that the cytoplasmic extract clearly supplied other additional missing fractions. E. coli DNA polymerase I, together with ligase, gave only a small stimulation of the overall synthesis, but increased the joining to a larger extent than ligase alone. Both the polymerizing activity and the exonuclease activity of this enzyme might be responsible for the observed effect. The experiments involving addition of highly purified enzymes indicate that proteins with molecular weights of about 100,000 (6, 13) can influence a synthetic process which apparently occurs inside nuclei. It seems reasonable to assume that this occurs after penetration of the enzyme into the nuclei. The nuclear system was originally developed with the hope that it would be useful in the dissection of intermediate steps of polyoma DNA synthesis. The effects described in this paper open up experimental possibilities in that direction. The demonstration of stimulatory effects on a depleted system must be interpreted with great caution since clearly neither E. coli ligase nor E. coli DNA polymerase I per se participate in polyoma DNA synthesis. The demonstrated permeability for proteins of the nuclear in vitro system appears promising, however, and it now seems experimentally feasible to identify enzymes participating in the intermediate steps of DNA synthesis either by fractionation of cytoplasmic extracts or by immunological techniques.

tracts from infected

267

ACKNOWLEDGMENTS We thank Gunilla S6derman for expert assistance. The study was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft to B. 0. and by grants from the Swedish Cancer Society and Magnus Bergvalls Stiftelse to P. R. LITERATURE CITED 1. Crawford, L. V., C. Syrett, and A. Wilde. 1973. The

replication of polyoma DNA. J. Gen. Virol. 21:515-521. 2. Eliasson, R., R. Martin, and P. Reichard. 1974. Characterization of the RNA initiating the discontinuous synthesis of polyoma DNA. Biochem. Biophys. Res. Commun. 59:307-313. 3. Francke, B., and T. Hunter. 1974. In vitro polyoma DNA synthesis: discontinuous chain growth. J. Mol. Biol. 83:99-121. 4. Hershey, H. V., J. F. Stieber, and G. C. Mueller. 1973. DNA synthesis in isolated HeLa nuclei. Eur. J. Biochem. 34:383-394. 5. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. 6. Jovin, T. M., P. T. Englund, and L. L. Bertsch. 1969. Enzymatic synthesis of DNA. XXVI. Physical and chemical studies of a homogenous DNA polymerase. J. Biol. Chem. 244:2996-3008. 7. Klein, A., and F. Bonhoeffer. 1972. DNA replication. Annu. Rev. Biochem. 41:301-332. 8. Magnusson, G., V. Pigiet, E.-L. Winnacker, R. Abrams, and P. Reichard. 1973. RNA linked short DNA fragments during polyoma replication. Proc. Nat. Acad. Sci. U.S.A. 70:412-415. 9. Magnusson, G., E.-L. Winnacker, R. Eliasson, and P. Reichard. 1972. Replication of polyoma DNA in isolated nuclei. II. Evidence for semiconservative replication. J. Mol. Biol. 72:539-552. 10. Mueller, G. C. 1969. Biochemical events in the animal cell cycles. Fed. Proc. 28:1780-1789. 11. Okazaki, R., T. Okazaki, K. Sakabe, K. Sugimoto, R. Kainuma, A. Sugino, and N. Ivatsuki. 1968. In vivo mechanism of DNA chain growth. Cold Spring Harbor Symp. Quant. Biol. 23:129-143. 12. Olivera, B. M., and I. R. Lehman. 1967. Linkage of polynucleotides through phosphodiester bonds by an enzyme from Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 57:1426-1433. 13. Soderhiill, S., and T. Lindahl. 1973. Two DNA ligase activities from calf thymus. Biochem. Biophys. Res. Commun. 53:910-916. 14. Westergaard, O., D. Brutlag, and A. Kornberg. 1972. Initiation of DNA synthesis. IV. Incorporation of the RNA primer into the phage replicative form. J. Biol. Chem. 248:1361-1364. 15. Winnacker, E.-L., G. Magnusson, and P. Reichard. 1972. Replication of polyoma DNA in isolated nuclei. I. Characterization of the system from mouse fibroblast 3T6 cells. J. Mol. Biol. 72:523-537.

Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

JOURNAL OF VIROLOGY, Feb. 1975, p. 259-267 Copyright @ 1975 American Society for Microbiology Vol. 15, No. 2 Printed in U.S.A. Replication of Polyom...
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