DNA replication in SV40-infected cells

VIROI‘QGY

(1972)

48,373-379

NA

Replication

LIThe Effect of Cycloheximide RUDOLF Department

in on the Formation of

JAENISCH

of Biochemistry,

Princeton

AND ARNOLD University,

J. LEVINE

Princeton,

New Jersey 08540

Accepted January IY, 1972 The effect of cycloheximide on the form&ion of SY40 oligomeric DNA was examined. About 1.5yc of the total viral DNA synthesized in an infected cell sedimented as SV40 dimers while 0.2% of the total DNA was found astetramers. The addition of cycloheximide to these cells resulted in a 70-90% decrease in the formation of SV40 monomeric DNA, but little or no decrease in the formation of SV4O dimers and tetramers. Thus, SV40-specific dimers (3-6%) and tetramers (0.5-1.5’%) represented a larger percentageof the total viral DNA synthesizedin the presence of cycloheximide. Viral DNA synthesized prior to the addition of cycloheximide did not participate, or participated to only a small extent, in the formation of SV40 oligomeric DNA observed after cycloheximide treatment. These results demonstrate that SV40 oligomers were not formed by recombination of monomeric viral DNA synthesized prior to cycloheximide treatment. The available evidence indicates that SV40 oligomers were synthesized by the replicat,ion of viral DNA in the absence of protein synthesis. INTRODUCTIQX

(1968) and Goebel (1971) have shown that oligomeric forms of circular DNA from the Oligomerie forms of covalently closed bacterial plasmid coheine E1 can be gener. circular DNA have been isolated from a ated by inhibition of protein synthesis. The> variety of sources (Rush and Warner, 1968; propose that the formation of ohgomersmay Rhoades and Thomas, 1968; Hudson and result from an imbalance in the levels of Vinograd, 1967; Jaenisch et al., 1969; Nass, enzymes responsible for cohcine El DKA 1969; Cuzin et al., 1970). Recently SV40replication, In a similar ma,nner the op specific circular and catenated ohgomer posing rolling circle model of DressHer and were detected during SV40 lytic infection Wolfson (1970) and t,he Yoshikawa of monkey cells (Rush et al., 1971; Jaenisch model for Bacillus subtilis DNA repli Two quite different mechanismshave been would explain the formation of circu%nr oligomers as intermediates in the replication proposed to explain the formation of oligomers from monomeric forms of circular process. The experiments presented in t per DNA. Hudson and Vinograd (1967) have were initiated in an effort to dete (I) outlined a scheme where recombination whether oligomeric forms of SV40 are events between circular molecules lead to the generated at an enhanced rate when proteii: formation of circular and cat8enated oligomers. The investigation of Rush and Warner synthesis is inhibited; and (2) whether these (1968) on the formation of +X174 dimers oligomers arise by recombinat(ion or ‘DKA repliMion. during infection of Escherichia coli with temperature-sensitive mutants of +X174 indeed demonstrates the occurrence of such Virus. The SV4O small-plaque virus was er=recombination events. On the other hand! ployed throughoilt. these experiments, This virus the experiments of Goebel and Helinskl was kindly supplied by Dr, M. Martin. 373 Copyright

0 1972 by Academic

Press,

Inc.

374

JAENISCH

Tissue culture. Monolayers of primary and secondary African green monkey cells (AGMK) (Flow Laboratories) were grown as described by Levine et al. (1970). Preparation of virus stocks. Monolayer cultures of AGMK cells were infected at an input multiplicity of 2-5 PFU/cell and harvested as previously described (Levine et al. 1970). Isotope labeling and extraction of nucleic acids. Monolayer cultures of AGMK cells were infected with XV40 at an input multiplicity of 69-80 PFU per cell. Fourteen hours after infection, 1 mCi of a2P (inorganic phosphate, Mann-Schwartz) was added to each petri dish (15 ml of medium). For pulse labeling with 3H-thymidine, the cultures prelabeled with a2P were washed twice with prewarmed medium, and 4 ml of medium containing 0.5 or 1.0 mCi of 3H-thymidine (23 Ci/mmole, Mann-Schwartz) was added. When DNA synthesis was examined in the presence of eycIoheximide, 15 or 20 pg/ml of this drug (Sigma) was added. This level of cycloheximide is sufficient to inhibit 96-99y0 of the incorporation of 3H-leucine into trichloroacetic acid-precipitable counts (Kang et al., 1971). The nucleic acids were extracted as described by Jaenisch and Levine (1971a). Centrifugation techniques. Alkaline sucrose gradient centrifugation was performed by sedimenting the nucleic acids through a lo-3Oyn linear sucrose gradient (total volume, 12 ml) using a Spinco SW 41 Ti rotor. The sucrose stock solutions were mixed shortly before use with alkaline buffer according to Jaenisch and Levine (1971a). To obtain sucrose solutions with a pH of 12.2, the following solutions were mixed and always kept at 4”: 10 ml of 609;’ sucrose containing 0.3 &f NaCl and 2 X low3 M EDTA were mixed with 6 ml of distilled water, 0.3 ml of 2 M glycine-NaCl solution, and 3.7 ml of 2 M NaOH to give a 30y0 sucrose solution; 10 ml of 20% sucrose containing 0.3 M NaCl and 2 X 10-3iw EDTA were mixed with 6 ml of distilled water, 1.335 ml of 2 M glyeineNaCl solution, and 2.58 ml of 2 M NaOH to give a 10% sucrose solution. Centrifugation was performed at, 39,000 rpm and 6” for 6 or 7.5 hr. Fractions were collected by puncturing a hole in the bottom of the tube and tested for radioactivity. The desired portions of the gradient were pooled, neutralized, and concentrated by precipitation with ethanol. Alkaline CsCl gradients were prepared by centrifugation of 5 ml of an alkaline C&l solution (density 1.3 g/ml in 0.1 AT N&H, 2 X 1O-3 M EDTA) at 40,000 rpm for 5 hr at 15” in a Spinco SW 50.1 rotor. A 0.15.ml sample containing nucleic acids in 0.1 N NaOH and 2 x 10-B M EDTA was layered on top of the preformed

AND

LEVINE

gradient and centrifuged for 45-60 min at 35,000 rpm. Samples were collected and tested for radioactivity as described (Levine et al., 1970). RESULTS

Isolation of SV40 Oligomers Formed in the Presenceor Absence of Cycloheximide The goal of the following experiments was to investigate the effect of cycloheximide on the formation of SV40 oligomeric DNA. Since oligomers formed in the presence of cycloheximide were to be compared with molecules synthesized prior to the addition of the drug, a double labeling procedure was employed. Confluent monolayer cultures of AGMK cells were infected with SV40 as described previously, and 32P-labeled inorganic phosphate was added to the cultures at 14 hr after infection to label uniformly all nucleic acids synthesized. At 38 hr post infection the cultures were washed twice and 3H-thymidine was added in the presence or absence of 15 pg/ml of cycloheximide. Two hours later the nucleic acids were extracted and analyzed by sedimentation through an alkaline sucrose gradient. In alkaline sucrose (pH 12.2), covalently closed double-stranded circular molecules sediment in a cyclic coil configuration with a high sedimentation coefficient whereas relaxed circular molecules are denatured and sediment more slowly. As can be seen in Fig. 1, two major peaks of 32Pand 3H cpm were observed in the alkaline sucrose gradient. The faster sedimenting peak contains ’ SV40 monomeric closed circular DNA, and the slower sedimenting peak corresponds to the position in the gradient expected for relaxed SV40 DNA, some mitochondrial DNA, and low molecular weight cellular DNA (Saenisch and Levine, 1971a). The large peak of 32P-radioactivity at the top of the gradient is due to degraded labeled RNA. SV40 oligomeric DNA is expected to sediment as a shoulder on the leading edge of the peak of monomeric DNA (Jaenisch and Levine, 1971a). In order to accurately detect the level of SV40 oligomers synthesized, the fractions indicated by the bar in Fig. 1 were pooled, concentrated and further analyzed by sedimentation through alkaline CsCl (Fig. 2A and B).

CYCLOHEXAMIDE

AND

XV40

OLIGOMERIC

‘,P*) i 2

DiYA

amount of DKA synthesiz;ed). The 33 :V ratio remains roughly constant (0.75--I 2.1) in the oiigomeric regions of the gradient for SV40 DNA synthesized in the absence ni’ cycloheximide (Fig. ‘.?A). This indicates t#h,n,t

20 FRACTION

40

30

NUMBER

FIG. 1. Sedimentation of nucleic acids from SV4Q-infected AGMK cells through an alkaline sucrose gradient. Two infected AGMK monolayer cultures were iabeled with 32P from 14 to 36 hr p.i. The cells were washed twice and labeled with 0.5 mCi %I-thymidine in the absence (A) or presence (43) of 15 fig/ml cyeloheximide for 2 additional hours. The nucleic acids were extracted and sedimented through a 19-307, alkaline sucrose gradient for 6.5 hr at 39,000 rpm in a SW 41 Ti rotor. The fractions obtained were tested for radioactivity. The bars indicate fractions which were pooled and further a,nalyzed. X--X, 3H cpm; O--O, 32P cpm.

In the alkaline CsCl gradient, two major peaks of radioactivity were resolved. These two major components sedimented in the position expected for SV40 monomers and dimers. Smaller levels of radioactivity sedimented asexpected for trimers and tetramers (seefor eomparison and further charact,erization of t,hese components, Jaenisch and Levine, 1971a). In t’he upper portion of Fig. 2A and B t,hc 3R : 32Pr&o of each fraction is plotted. The 3R:32P ratio at the position of SV40 monomers was normalized to one. This allows the detection of any differences in the percentage of oligomers produced in the presence or absence of cycloheximide (the amount

of oligomers

synthesized/total

10

20 FRACTION

30 NUMBER

FIG. 2. Sediment,ation of SV4O DNA through alkaline CsCl. Labeled nucleic acids obtained from the gradients in Fig. 1 (indicated by bars in Figs 1) were sedimented through alkaline CsCl at 35,000 rpm for 45 min in a SW 50.1 rotor. An alkaliue CsCX solution (1.3 g/ml) was centrifuged for 5 hr at 40,000 rpm to preform a gradient for sedimentsing the nucleic acids. Fractions were collected and tested for radioactivity. In the upper parts of the graphs the %I: 32P ratio of each fraction is plotted. The values are normalized to t,he rat,io found at tbe SV40 monomeric peak (fraction 27) in each gradient, which was set as 1. {A) IYucleic acids from gradient in Fig. 1A. (B) Nucleic acids from gradient in Fig. 1B. X--X, % cpm; a---n %I:a2P ratio.

376

JAENISCH

AND

the percentage of SV40 oligomeric DNA made during a 2-hr pulse labeling with 3Hthymidine at 36-38 hr after infection was similar to the percentage of oligomers formed during 14-36 hr after infection. On the other hand, the SV40 DNA synthesized in the presence of cycloheximide (Fig. 2B) was converted to oligomeric forms of DNA at a higher rate relative to the levels of monomers synthesized (3H:32P ratio of oligomers was 2-2.5). In order to quantitate the fraction of SV40 DN-4 found as oligomers in the presence or absence of cycloheximide, the levels of radioactivity found in the peaks of SV40 monomers in Figs. 1 and 2 and for SV40 dimers and tetramers in Fig. 2 were computed. These data are presented in Table 1, experiment 1. It can be seen that there was a 70 % decrease in the incorporation of 3H-thymidine into total viral DNA over the first 2 hrs of cycloheximide treatment. At the same time there was no decrease in the formation of SV40 dimers and tetramers. This dif-

LEVINE

ferential inhibition of viral DNA synthesis resulted in a 3-fold increase in the pcrcentage of 3H-label found in dimers and tetramers synthesized in the presence of cycloheximide. If this relative increase in the percentage of oligomers formed was due to recombination of monomers synthesized prior to the cycloheximide treatment (and therefore 32P-labeled) a similar increase in the percentage of dimers and tetramers that were 32P-labeled would be expected during the cyeloheximide treatment. The 32P-labeled oligomers only increased 1.4-fold. The fact that there is an increase in 32P-labeled oligomers at all is thought to be due to the inability to completely chase the 32P-label (found in RNA and in the pools). When 3H-thymidine was employed prior to cycIoheximide treatment (instead of 32P) an efficient chase in the presence or absence of cycloheximide was obtained. Under these circumstances, no increase in the percentage of 3H-labeled dimers was observed after the addition of cycloheximide.

TABLE QUANTITATION

Labeling

OF SV40

period

Total cpm found in closed circular viral DNA

with

Expt. No. 32P (hr p.i)

1

14-36

14-36

2

(h:t.ij

36-38

36-38

1

OLIGOMERIC DNA FORMED OF CYCLOHEXIMIDE~

Cyclohex. present

36-38

-

IN THE PRESENCE

y0 labeled

viral

OB ABSENCE

DNA

found

Dimers 32P

130,000

133,000

in

Tetramers

3H

48,000

137,000

14-38

4042

38-42

52,500

36,500

14-38

4042

-

62,000

360,000

32P

3H

1.55 (2000)

0.2

0.5

&";O,

(260)

(240)

1.05 (1400)

1.0 (1370)

(lf36)

(206)

6.0 (2200)

0.6 (310)

1.45 (530)

0.38 c:fZO)

(240)

0.33 (1150)

1.4 (870)

32P

0.14

3H

0.15

a Confluent. AGMK cells were infected and labeled in the presence or absence of cycloheximide as described in Materials and Methods. The DNA was extracted and analyzed on alkaline sucrose and alkaline CsCl gradients as described in Figs. 1 and 2. The radioactivity in virus-specific DNA of both gradients was computed, and the results are expressed as the percentage of radioactivity found in oligomeric DNA of the total radioactivity incorporated into viral supercoiled DNA. The numbers in parentheses represent the total counts per minute found in dimers and tetramers.

CPCLOHEXAhfIDE

AND

XV40 Oligomers Represent an Increasingly Larger Percentage of the Total Viral DNA Synthesized with Increasing Time of Cyclohezimide Treatment The previous experiment, demonstrated a 3-fold relative increase in the percentage of SV40 oligomers synthesized when protein synthesis was inhibited. The synthesis of SV4Q DNA is not completely stopped aft,er addition of cycioheximide but continues to decline with mcreasing time of exposure to this drug (Iiang et al., 1971). It was of interest to seewhether the enhanced relative rate of dimer formation would continue t,o increase as the ra,te of viral DXA symhesis decreased. Two monolayer cultures of AGMK cells were infected with SV40 and labeled with 32P-inorganic phosphat,e from 14 to 38 hr. after infection. These cultures were washed, and eycloheximide was added to one of t.hem. Two hours later (40 hr after infection) 3Hthymidine was added to both cultures for a 2-hr labeling period. At 42 hr post infection both cultures were harvested, and the nucleic acids lvere sedimented through alkaline sucrose and alkaline C&l gradients to analyze the SV4Q oligomeric DNA as described above. The results of this experiment are presented in Table 1, experiment 2. It can be seenthat there was about a 5-fold increase in the percentage of 3H-labeled DKA that sedimented like SV40 dimers and tetramers. A comparison of the experimental results presented in Table 1 shows that l-l.5 % of the viral DNA could be isolated as SV40 dimers in untreated cells. The viral DNA synthesized during the first 2 hr of cycleheximide inhibition contained about 3% dimer molecules. The viral DNA synthesized during 2-4 hr after the addition of eycloheximide contained about 6 % dimerie SV40 DNA. A similar relationship holds for SV40 tetramerie DNA. Thus, SV40 dimers and tetramers represent an increasingly larger percentage of the total viral DNA synthesized with increasing time of cycioheximide inhibition. The Effect of Mitochondrial DNA Analysis of SV40 Oligomers

on the

Since mitochondrial DNA has a molecular weight in the range of SV40 trimers (Jaenisch

SV40 OLIGOMERIC

DNA

37i

and Levine, 197La) it was important TV eliminate ‘the possibilit,y that SV40 oligomers were contaminated with mitoehondria,! DNA. In the procedure used here to isolat,e SV40 closed circular pH 12.2, followed by 13.0) almost all the mitochondrin~ DNA is denatured to single strands while SV40 IPSA is relatively resi&anL To show t chondrial DXA was labeled with dine and isolated as described (1971). This DKA was sedimented v&h Ww labeled SV4O DNA through an alkaline C&I gradient. During this procedure virtuailg all mit’ocholndrial DNA (greater than 98 %) W:E denatured to a single-stranded form and sedimented more slowly than SV4O monomeric DNA. Thus mitochondrial DNA did not contribute to the analysis of oligomerk SV40 DT\‘A. DISCCSSIOX

The addition of eycloheximide to SV40 infected AGMK cells resulted in inhibition in the incorporation of dine into monomeric viral D same culls the format,ion of and tetrameric molecules was not inhibited or was inhibited to a much smaller extent. Thus SV40 oligomers made in the presence of cycloheximide represented a much larger proportion (3- to &fold larger) of the t’otnl viral DiYA synthesized after prowin synthesis was inhibited. Viral DNA synt,hesized prior to cycloheximide treatment did not contribute, or contributed to a much smaller ext,ent, to the formation of SV40 oiigomerio DNA generated 6n the presence of cycl~eheximide. Hit and Nakajima (1971) failed t.o detect the formation of $8740oligomerie DSA after the addition of cyeioheximide to SV4Oinfected CV-1 cells. It appears likely that the methods they employed were no+Lsensitive enough to detect SV4O dimers oecurrmg at a l-6 % level. at least t\+ro Qays t,hat SVB.? NA could be formed in the presence of cycloheximide: (I) genetic recombination of viral monomers that were synthesized aft,er the addition of cy&heximide and (2) the replicat,ion of viral monomeric DYA in the absence of protein

378

JAENISCH

synthesis occasionally yielded a dimer instead of two monomers. Several lines of evidence favor, but do not prove, the latter alternative. First, SV40 tetramers are reproducibly found in larger quantities than are SV40 trimers (see Fig. 2 of this paper and Jaenisch and Levine, 1971a). One might expect that a monomer (94-97 % of the total DNA made in cycloheximide) and a dimer would recombine to give a trimer more frequently than two dimers (or a trimer and monomer) would recombine to yield a t,et)ramer. On the other hand, once a monomer replicated to yield a dimer, the dimer could rep1icat.e to yield a tetramer. The fact that SV40 dimers can replicate has been shown previously (Jaenisch and Levine, 1971b). Thus one would predict more tetramers than trimers should be present if oligomeric SV40 DNA was formed by an aberrant mode of DNA replication. Second, the experiments presented here have shown that cycloheximide inhibits the synthesis of SV40 monomers to a greater extent than SV40 oligomers. If oligomers arose bv recombination, a smaller pool of viral monomers made in the presence of cycloheximide must generate 3- to 5-fold higher levels of oligomers than in the untreated infected cell. This inverse relationship between pool size of viral DNA produced in the presence of cycloheximide and the formation of oligomers favors a DNA replication mechanism for the generation of SV40 dimers and tetramers. It is possible that oligomers are synthesized by a DNA replication process that does not require concurrent protein synthesis and is therefore distinct from the normal mode of SV40 monomer replication. Last, recent experiments (Jaenisch and Levine, in preparation) undertaken to characterize the type of dimers (circular or catenated dimers) formed in the presence or absence of cycloheximide show that the fraction of catenated dimers increases appreciably when DNA replication takes place in the absence of protein synthesis. The majority of SV40 replicating molecules isolated from infected cells contain closed circular templates (Jaenisch et cd., 1971;

AND

LEVINE

Sebring et al., 1971). Thus SV40 may require a nuclease-like action to separate each interlocked daughter replicated molecule. If this nuclease is inhibited by cycloheximide, a catenated dimer might result. ACKNOWLEDGMENTS We wish to thank A. Teresky for technical assistance and the Whitehall Foundation for general support. This research was supported by American Cancer Society Grant E-591, National Cancer Institute Grants CA11049-04, CA12968-01, and an International Fellowship of the National Institutes of Health (I-F05-TW 169901). REFERENCES CUZIN, F., VOGT, M., DIECKMANN, M., and BERG, P. (1970). Induction of virus multiplication in 3T3 cells transformed by a thermosensitive mutant of polyoma virus. II. Formation of oligomerit polyoma DNA molecules. J. Mol. Biol. 47,317-333. DRESSLER, D., and WOLFSON, D. (1970). In Watson, “The Molecular Biology of the Gene” (Watson, ed.), 2nd ed., p. 291. New York. GOEBEL, W. (1971). Formation of complex Co1 El DNA by replication. Biochim. Biophys. Acta 232, 32-42. GOEBEL, W., and HISLINSKI, D. (1968). Generation of higher multiple circular DNA forms in bacteria. Proc. Nat. Acad. Sci. U. S. 61, 14OtS 1413. HUDSON, B., and VINOGRAD, J. (1967). Catenated circular DNA molecules in HeLa cell mitochondria. Nature (London) 216, 647-652. JAENISCH, R., and LEVINE, A. (1971a). DNA replication in SV40 infected cells. V. Circular and catenated oligomers of SV40 DNA. virology 44, 480-493. JAENISCH, R., and LEVINE, A. J. (1971b). Infection of primary African green monkey cells with SV40 monomeric and dimeric DNA. J. Mol. Biol. 61, 735-738. JAENISCH, R., HOFSCHNEIDER, P. H., and PREUSS, A. (1969). Isolation of circular DNA by zonal centrifugation. Separation of normal length, double length and catenated Ml3 replicative form DNA and of host specific episomal DNA. Biochim. Biophys. Acta 190, 88-100. JAENISCH, R., MAYER, A., and LEVINE, A. J. (1971). Replicating SV40 molecules containing closed circular template DNA strands. Nature New Biol. 223, 72-75. KANG, S., ESHBACH, D., WEITE, D., and LEVINE, A. (1971). DNA replication in SV40 infected cells. IV. Two different requirements for pro-

CYCLOHEXAMIDE

AND

rein synthesis during SV40 DNA replication. J. Viral. 7, 112-120. KIT, S., and NAKAJIMA, K. (1971). Analysis of the molecular forms of SV40 DNA synthesized in cycloheximide treated cell cultures. J. Viral. 7, 87-94. LEVINE, A. J. (1971). DNA replication in SV40 infected cells. The induction of mitochondrial DKA synthesis in monkey cells infected by SV40 or treated with calf serum. Proc. Nat. Acad. Sci. LT.;.S. 68, 717-720. LEVINE, A. J., KANG, H. S., and BILLHEIMER, F. (1970). DNA replication in SV40 infected cells. 1. Analysis of replicating SV40 DNA. J. &loZ. BioE. 50, 549-568. Nass, M, (1969). Reversible generation of circular dimer and higher multiple forms of mitochondrial DNA. Nature (Lo&on) 223, 112+&1129.

SV40

OLTGOMERIC

DI\iA

379

and Tao~as, 6. (1968). The P22 DNA molecule. II. Circular informs. J. ~Wol. Biol. 37, 41-61. RUSH, M., and W.ARNER, R. (1968). Multiple length rings of +X174 and S13 replicative forms. III. B possible intermediate in recombination. J. Biob. Chem. 243, 4821-4826. RUSH, M., EASON, R., and VIXOGEAD, j, (1971). Ident’ification and properties of complex forms of SV40 DNA isolated from SV40 infected cells. Biochim. Biophys. Beta 228, 585-594. RHOADES,

M.,

bacteriophage tracellular

SEBRING,

E.

I>.,

KELLY,

T.

J.,

N. P. (1971). Structure simian virus 40 deoxyribonucleic J. viroz. 8, 478-490. Yosrrrr~~va, H. (1967). Initiat.ion cation in Bacillus subfilis. Proc. u. s. 58, 312-319. SALZMAN,

THOXEN,

M,

M.$

of replicating acid moleeul~ss. of DKA repliNat. Acud. SC<.