JOURNAL OF VIROLOGY, Apr. 1976, p. 307-315 Copyright © 1976 American Society for Microbiology
Vol. 18, No. 1 Printed in U-SA.
Bidirectional Replication of Adenovirus Type 2 DNA MARSHALL S. HORWITZ Department ofMicrobiology-Immunology, Cell Biology, and Pediatrics, Albert Einstein College ofMedicine, Bronx, New York 10461 Received for publication 11 November 1975
After short periods of labeling with [3H]thymidine, recently completed adenovirus DNA molecules were isolated and cleaved with restriction endonucleases. The strands (heavy and light) of most of the restriction endonuclease fragments were separated. The pattern of labeling clearly shows an asymmetry of radioactivity on the isolated strands of each restriction endonuclease piece. The data is consistent with replication proceeding in the 5' to 3' direction on each strand. Thus, there is an initiation point placed at or near each end of the molecule. Adenovirus type 2 DNA, a linear molecule of 23 x 106 daltons, replicates in the nucleus and produces approximately 100,000 copies/cell during lytic infection (9, 10). The duplex DNA has nonpermuted sequences (6) and inverted terminal redundancy at ends (8, 34), which are identical for 100 to 140 nucleotide pairs (1). The inverted terminal redundancy allows the formation of single-strand (ss) circles by base pairing between both ends ofthe denatured ss molecules. Since the ends of adenovirus duplex DNA are identical, these molecules cannot be converted to covalently linked double-strand (ds)
circles like those formed by bacteriophage lambda DNA (10, 34). During replication, ss molecules larger than the genome have not been demonstrated (12, 29, 30); therefore, no covalent addition of progeny DNA to parental molecules occurs. There are small pieces of adenovirus DNA similar to "Okazaki fragments" (20), which can be dissociated from the replication complex by alkaline denaturation (2, 12, 31, 33). During normal viral replication it is not known if all regions of the genome are first polymerized into Okazaki fragments, which are subsequently joined. Replicating molecules have a significantly higher buoyant density than parental viral DNA (27, 29). Further evidence has shown that the density shift is caused by extensive ss DNA regions and not by RNAprimer fragments as reported for polyoma DNA replication (16). Recently, a number of models of replication, which include bidirectional growth, have been proposed for adenovirus DNA. Sussenbach and co-workers have presented data that replication starts at the right end (AT rich) by displacing the parental heavy strand with continuous polymerization in the 5' to 3' direction on the light-strand template (7). After a delay, replication starts on the displaced strand at various internal points or may
start at the 3' end of the template molecule in a pattern of continuous growth in the opposite direction to that on the first strand. Experiments from our laboratory, in which adenovirus DNA half-molecules were produced by mechanical shearing, have shown that there is bidirectional growth with two termination sites, one on the left and another on the right half of the molecule (13). The present study extends these findings by using two restriction endonucleases, which have allowed us to examine nine regions of the DNA. The method to determine the origin and terminus of DNA replication is similar to that employed by Dintzis (5) to analyze the direction of replication of polypeptides and more recently by Danna and Nathans (4) to isolate the origin and direction of replication of simian virus 40 DNA. When a radioactive precursor is added to a system synthesizing DNA, the label enters replicating molecules at a growing point that is at a different site in each molecule. This assumes that the labeling procedure does not change the rate of DNA synthesis by synchronizing molecules at any phase of the replication cycle. The molecules, which are completed during labeling periods shorter than the total synthesis time of the macromolecule, will be preferentially labeled at the terminus. Therefore, when completed molecules are effectively separated from replicating molecules, the amount of radioactivity in various regions of the completed molecules will reflect the origin and direction of synthesis. In this report the pattern of labeling of ds DNA indicates highest specific activity at both ends of the DNA, which is consistent with a termination site at each end of the molecule. While this manuscript was in preparation, similar results were reported (23, 28). However, examination of the single-strands 307
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of each of these DNA fragments has shown that the heavy and light strands label asynchronously. This difference in labeling is most consistent with replication in which each strand is synthesized in the 5' to 3' direction starting at opposite ends of the molecule. (A preliminary report of these results was presented at the Cold Spring Harbor Tumor Virus Meeting, Cold Spring Harbor, N. Y., August, 1975.) MATERIALS AND METHODS Cells and viruses. The source of HeLa cells, adenovirus type 2, and the conditions of infection have been previously described (17). All experiments were done in suspension cultures at an input multiplicity of 4,000 virions/cell (100 to 200 PFU/cell). Radioactive labeling of cells. At 18 h postinfection, the infected HeLa cells were centrifuged for 2 min at 1,500 rpm in an International PRJ centrifuge and resuspended at a concentration of 107 cells/ml in Eagle spinner medium with 5% fetal calf serum. After 5 min of temperature equilibration in a water bath at 37 C, the cells were radioactively labeled with [3H]thymidine at 0.25 mCi/ml (40 to 60 Ci/ mmol). The incorporation of radioactivity was stopped by diluting the cells in 7 volumes of ice-cold Earle salts, rapidly centrifuging the cells at 1,500 rpm, and adding 0.2% sodium dodecyl sulfate in 0.01 M Tris-EDTA (pH 7.4). Using these conditions, the incorporation of radioactivity was linear for 60 min without any appreciable lag at the beginning of this interval (14). Purification of viral DNA. Intact [3H]thymidinelabeled viral DNA was purified from cells after precipitating large-molecular-weight cell DNA by a modification of the Hirt procedure (11, 29, 30). Cells (3 x 107) were suspended in 2 ml of 0.01 M Tris, 0.01 M EDTA (pH 7.4) at 0 C. Sodium dodecyl sulfate (0.2%) and 500 ,ug of Pronase (preincubated at 37 C for 2 h to digest any residual nucleases) were added, and the mixture was incubated for 15 min at 30 C. The volume was increased to 9 ml by the addition of the Tris-EDTA buffer, which contained 1% sodium dodecyl sulfate. After a 5-min incubation at 30 C, NaCl was added to a final concentration of 1 M. The solution was left at 4 C for 16 h, and the precipitate was removed by centrifugation at 12,000 rpm for 20 min in a Spinco angle 30 rotor. By processing 1.5 x 107 to 3 x 107 cells in a final volume of 10 ml, 80% of newly replicated adenovirus DNA was recovered in the Hirt supernatant. Viral DNA was precipitated from the supernatant with 2 volumes of ethanol. The DNA, redissolved in 1 ml of 0.01 x SSC (SSC = 0.15 M NaCl + 0.015 M sodium citrate), was centrifuged in an SW27 rotor of the Spinco ultracentrifuge (16 h at 22,000 rpm) on 16-ml, 5 to 20% neutral sucrose gradients containing 1 M NaCl, 0.01 M phosphate buffer, and 0.01 M EDTA. The 31S fractions were pooled, dialyzed against 0.3 M NaCl, 0.01 M Tris, 0.01 M EDTA (pH 8.1), and loaded onto 2-ml columns of benzoyl-naphthoyl-DEAE-cellulose (BND-
J. VIROL. cellulose). The ds DNA was eluted with 1 M NaCl, and the DNA containing any ss region was eluted with 1 M NaCl and 2% caffeine in the same TrisEDTA buffer (29). The appropriate column fractions were precipitated with 2 volumes of ethanol and redissolved in 0.01 x SSC. ['4C]thymidine-labeled viral DNA, which was used as a uniformly labeled marker, was purified by disrupting virion which had been banded twice on CsCl density gradients (12). Restriction endonucleases. The enzymes from both Escherichia coli (EcoRI) and Haemophilus parainfluenzae (HpaI) were purified from bacterial strains obtained from the Cold Spring Harbor Laboratory. The bacteria were grown and the enzymes were purified as previously described (19, 25). All endonuclease digests were incubated for 4 h at 37 C in 10 mM Tris-hydrochloride (pH 7.4) with 10 mM MgCl2, 6 mM KCl, and 1 mM dithiothreitol. The reaction was stopped in 0.04 M EDTA; the solution was adjusted to a final concentration of 10% sucrose and 0.1% bromophenol blue. The DNA fragments were separated by electrophoresis on cylindrical gels (1.6 by 35 cm) of 1.4% agarose in Tris-EDTA-acetate (TEA = 40 mM Tris-hydrochloride, 1 mM EDTA, 5 mM sodium acetate) buffer for 16 h at 100 V. The gels were stained in the same TEA buffer containing 0.5 ,ug of ethidium bromide per ml, and the bands were visualized with a UV-light source (25). For separation of the strands of each restriction endonuclease fragment of DNA, the ds fragments were cut out of the agarose gel after staining with ethidium bromide and visualization by minimal exposure to UV irradiation. The short cyclindrical piece of gel was placed into a glass scintillation vial with 10 ml of 0.2 M NaOH for 2.5 h at room temperature. The NaOH was decanted, and the gel was soaked for a further 2 h in TEA electrophoresis buffer at 0 C. The gel slice was placed back into the electrophoresis tube, and a new column of gel was polymerized over the original slice. The agarose was poured after equilibration at 60 C. After the polymerization, the gel tube was inverted, and the electrophoresis was performed under identical conditions as used for the original separation of the ds fragments. The gel were stained with ethidium bromide and the single strands were visualized. The separated strands were cut from the gel, and the radioactivity was counted. Radioactivity in the gel slices was quantitated by remelting the agarose in an autoclave and then adding 10 ml of scintillation fluid [1 part Triton, 2 parts toluene, 5 g of 2,5-diphenyloxazole (PPO) per liter, and 50 mg of 1,4-bis-(5-phenyloxazolyl)benzene (POPOP) per liter], which had been heated to 50 C. The samples, which were shaken immediately after the addition of scintillation fluid, were cooled to room temperature and counted in the ambient temperature scintillation counter (Beckman LS 230). Reagents. [3Hlthymidine (40 to 60 Ci/mmol) and ['4C]thymidine (57 mCi/mmol) were purchased from Schwarz BioResearch, Inc. Agarose was obtained from Sargent Welch Co. BND-cellulose was purchased from Serva.
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cules on BND-cellulose, which eliminates any DNA with ss regions. The results of BND-cellulose chromatography of the 5-, 10-, 15-, and 240min samples are shown in Fig. 2. The first peak to elute (fraction 2) contains the completely ds molecules, and the second peak (fraction 8) represents any molecules with ss regions. Greater than 99.3% of DNA, purified from virion and similarly treated, elutes in the ds region. When DNA from the virion is denatured by boiling before chromatography, 100% of the DNA subsequently elutes in fraction 8, the ss region (data not shown). With the shortest pulse times examined (5 min), most of the DNA from the 31S region of the gradient has some ss regions, which are on molecules that have just initiated replication. By 15 min, the 31S region of the gradient has more labeled ds than ss molecules, and by 4 h 76% of the labeled DNA is in ds molecules. Determination of specific activity of ds regions of pulse-labeled DNA. The ds[3H]- thymidine-labeled DNA from fraction 2 (Fig. 2) was mixed with uniformly labeled [14C]thymidinecontaining DNA isolated from the adenovirion. The DNAs were digested either with the restriction endonuclease EcoRI or a mixture of this enzyme and that derived from HpaI. The patterns of digestion for EcoRI and HpaI have ') t0 10 been elucidated (18). The pertinent restriction endonuclease pieces are drawn to scale on the abscissa of Fig. 4. The EcoRI pieces are designated in capital letters (B to F) and the 0. HpaI fragments are designated in lowercase letters (e, c, f, a). "C-" and "E-" refer to EcoRI fragments further digested by HpaI with the loss of approximately 1.5% of the genome. The results of a typical EcoRI + HpaI restriction endonuclease digest of the mixture of [3H]DNA pulse-labeled for 5 min and the [U'4C]viral DNA are shown in Fig. 3. Peaks with the higher ratios of 3H- to '4C-labeled DNA include "c," "C-," and "e," which are near both ends of the molecules (see Fig. 4). A summary I0 top WCTION NUMBER of results obtained from 5-, 10-, and 15-min FIG. 1. Sedimentation velocity gradients to sepa- pulse-labeled DNAs are shown in Fig. 4. The rate replicating from completed viral DNA. Pulse- lowest specific activity (3H/14C) is in the region labeled DNA isolated from the Hirt supernatant was of fractions "a" and "B" near the center of the centrifuged on neutral sucrose gradients as described molecule, and the specific activity increases toin Materials and Methods. The gradients were frac- ward both ends. As expected, differences in the tionated into 0.75-ml aliquots, and the radioactivity specific activity of the individual pieces are was determined by removing 20 pl from each fraction greatest for the 5-min digest, less at 10 min, for quantitating the trichloroacetic acid-precipitable absent by 15 min. DNA. Fractions in the region of 31S-completed viral and minor or of specific activity clearly shows This pattern DNA (fraction 14) were pooled as designated by the bar, and the DNA was dialyzed against 0.3 M NaCl, bidirectional growth of the molecule, but a 0.01 M EDTA, and 0.01 M Tris (pH 8.1) before number of quite different models could explain chromatography on BND-cellulose (Fig. 2). Symbols: this data. 0, 5-min pulse; 0, 15-min pulse. To differentiate between numerous possibiliRESULTS Isolation of recently completed viral DNA molecules. Eighteen hours postinfection, cells were radioactively labeled with [3H]thymidine and viral DNA was separated from host DNA by using the Hirt procedure. The Hirt supernatant, containing replicating and completed viral DNA molecules, was centrifuged on neutral sucrose gradients as shown in Fig. 1. The completed molecules (31S) sediment to the region in the gradient that peaks at fraction 14. The heterogeneous population of replicating molecules sediment faster and appear between fractions 1 and 14 (29). The completed molecules were further purified free of replicating mole-
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N U M B E R F R A C T IO N from completed viral DNA. Two-milliliter to replicating separate FIG. 2. BND-cellulose chromatography columns ofBND-cellulose were prepared in 2.5-ml disposable plastic syringes by wetting the cellulose in 0.3 M NaCl, 0.01 M EDTA, 0.01 M Tris buffer (pH 8.1) containing 20% ethanol. The ethanol was removed by centrifugation of the resin immediately after suspension of the cellulose. The BND was poured into the columns and extensively washed with the same buffer without ethanol until the optical density of the effluent at 260 nm was less than 0.05. The DNA, which had been dialyzed against the column buffer, was loaded onto the BND and washed with 12 ml of the same buffer. Elution of ds DNA was achieved with 12 ml of the column buffer to which NaCI had been added to a final concentration of 1 M. Aliquots (2 ml) were collected during the elation, and 50-pJ samples were removed for quantitation of the DNA in each fraction. The column was then washed with 12 ml of the latter buffer, to which 2% caffeine (designated by the arrow) had been added. The ss DNA elated after the addition of caffeine and was quantitated by taking 50-pl aliquots for radioactivity counting. The ds DNA, elated in fraction 2, was precipitated with 2 volumes of ethanol. (A) *, 5 min; 0, 10 min. (B) A, 15 min; A, 240 min.
ties, it was necessary to know if replication was similar on both strands. Sharp et al. (24) have shown that the denatured heavy (H) and light (L) strands of most of the restriction endonuclease fragments can be successfully separated on agarose gels. Using the alkaline denaturation technique for DNA embedded in agarose (see above), we have been able to separate H and L strands from the EcoRI "B." "C," "D," "E," and "F" pieces and the HpaI "e" and "c" pieces. Determination of specific activity of ss regions of pulse-labeled DNA. The ratio of 3H (pulse)- to 14C (uniformly)-labeled DNA was determined for the separated strands of each restriction endonuclease piece isolated from recently completed duplex DNA as described in Materials and Methods. The extent of strand separation for several of the fragments is shown
in Fig. 5. The strand separation of DNA denatured within the agarose gel is superior to denaturation in solution not only because of the relative speed of the former technique but also because of the decreased amount of renatured DNA found after electrophoresis. In most experiments, as in the one shown in Fig. 5, there is no renatured DNA detectable by ethidium bromide staining. The assignment of strand specificity (H or L) for the isolated strands follows the designation of Sharp et al. (24). The faster moving band for the EcoRI "B." "C," "D," "E," and "F" and the slower band for HpaI "e" and "c" (P. A. Sharp, personal communication) belong to the same strand, which is the heavy strand in alkaline CsCl gradients. The HpaI "a" fragment does not separate into H and L strands under any conditions tried thus far.
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F R AC T I 0 N N U M B E R FIG. 3. Agarose gel electrophoresis of a restriction endonuclease digest of the 5-min pulse-labeled DNA. The ds viral DNA from fraction 2 (Fig. 2), which was extracted from cells pulse-labeled for 5 min with [3H]thymidine, was mixed with ["4C]thymidine-containing DNA purified from adenovirions. The mixture was digested with EcoRI and HpaI for 4 h as described in Materials and Methods. After electrophoresis of the DNA on 1.4% agarose columns for 16 h at 100 V, the gel was stained with ethidium bromide and the DNA bands were located. The gel was sliced into aliquots starting with a cut between the 'a" and Sc" pieces to insure that these closely migrating fractions were in separate gel slices. The gels were melted, and the radioactivity was quantitated by scintillation counting. The designation of the restriction endonuclease fragments is as described in the text. The location of the fragments and their relative sizes are shown on the abscissa ofFig. 4. Fragments "E-" and "F' do not separate in this system.
In Fig. 6 and 7, a summary of the specific activities of the isolated strands is shown for pulse-labeling periods of 5 and 10 min, respectively. It is clear that the specific activity is quite different for the corresponding regions on both strands, and this suggests that the L strand (0) is replicating from left to right and the H strand (0) from right to left. Singlestrand data for the "e" fragment at the extreme left end of the molecule is not shown in the figures but is presented in Table 1. The high specific activity on the L strand of the "e" frag-. ment and the low specific activity on the H strand do not continue the trend of labeling derived from quantitating replication on the other 96% of the molecule. DISCUSSION The data is most consistent with bidirectional growth, which is asymmetrical on each
of the strands of adenovirus type 2 DNA. The light strand appears to initiate at or near the left hand end and replicate continuously to the right. This corresponds to growth in the 5' to 3' direction according to the data of Sharp et al., who assigned the 5' end of the heavy strand to the right (24). In contrast, the heavy strand initiates on the right and replicates toward the lefthand end. This model of replication does not require Okazaki fragments to successfully propagate either chain, although several investigators have reported the presence of these intermediates. There appears to be a discrepancy of labeling at the lefthand end of the molecule, which was detected by examining the HpaI "e" piece representing 4% of the DNA. The light "e" strand had more radioactivity than expected for an origin of replication. This could occur because the origin of replication contains nucleotides
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such as RNA serving a primer function. Since licating molecule is a circle, and that replicathe RNA would have to be excised and replaced tion continues beyond the molecular end to an with deoxynucleotides, this region may be la- extent of 4% of the genome. Although linear beled by thymidine with kinetics similar to a adenovirus DNA molecules have terminal reterminus. This would occur if the RNA were dundancy, it is of the inverted type, which does removed toward the end of the replication on that particular strand. 3 OrAnother possible explanation is that the rep1.2*
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FIG. 6. Order of labeling (5 min) of selected regions of ss adenovirus DNA. DNA was labeled with e c f a B F D E C [3H]thymidine for 5 min in adenovirus-infected cells RESTRICTION ENDONUCLEASE FRAGMENT at 18 h postinfection. The recently completed duplex FIG. 4. Order of labeling of selected regions of ds viral DNA was purified and digested either with adenovirus DNA. From data such as that in Fig. 3, EcoRI for fragments B, F, D, E, and C or with EcoRI ratios ofpulse-labeled [3H]DNA to uniformly labeled + HpaI for fragments e, c, f, and a; the DNA was ['4C]DNA were calculated. Included are the data electrophoresed on agarose gels as described in from the 5-, 10-, and 15-min pulse-labeling periods 3. Selected fragments were processed to separateFig. the obtained either from digests using EcoRI alone (frag- H and L strands according to the description in Fig. ments B, F, D, E, C) or together with the HpaI en- 5 and in Materials and Methods. The specific activity zyme (fragments e, c, f, a). This results from the of the pulse-labeled DNA (PH) in relation to unithree time points are normalized to give identical formly labeled DNA (14C) was determined and plotratios for the "e" fragment. Symbols: 0, 5 min; 0, ted in relation to its map position on the adenovirus 10 min; A, 15 min. chromosome. Symbols: *, L strand; 0, H strand.
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Hpo. e Hpiior FIG. 5. Strand separation of restriction endonuclease fragments. Pulse-labeled DNA ([3H]thymidine) was mixed with uniformly-labeled DNA (['4C]thymidine) and digested either with EcoRI orHpaI. The pieces were separated on agarose gels, stained, and cut from the gel. After soaking the gel slices in 0.2 M NaOH to denature the DNA, the endonuclease fragments were rerun on neutral agarose gels as described in Materials and Methods. The results of strand separation are shown for several of the fragments. Although the HpaI "a" piece does not separate into H and L strands, the HpaI "c" and the EcoRI "B" fragment separate into two bands. (For other fragments successfully separated by this technique, see text).
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the "e" heavy strand, which is lower than expected for its position next to the "c" fragment. Similar data is not yet available from the qS righthand end of the molecule. The HpaI "g" 20h fragment, which is 1.4% ofthe genome, is at the right end. Although the "g" band was visualized with ethidium bromide staining and apu peared in fraction 44 (Fig. 3), the "g" radioactivity was always superimposed on a background of counts in fractions 45 through 47, which 10 makes exact calculations difficult. We have not yet been able to separate the strands of the "g" fragment and are approaching the quantitation of counts on each of the strands by hybridiza05K tion of duplex "g" fragment with isolated strands of the EcoRI "C" fragment. It is also possible that labeling at the molecu1 IT lar ends may be complicated by an inability to f B F D E C c e separate newly initiated molecules with very RESTRICTION ENDONUCLEASE FRAGMENT FIG. 7. Order of labeling (10 min) of selected re- short replicating regions from the pool of ds, gions of ss adenovirus DNA. Infected cells were la- 31S completed molecules. It is difficult to debeled with [3H]thymidine for 10 min in the same sign a control experiment for this possibility. experiment as described in Fig. 6. The DNA was The models of adenovirus replication as a processed and quantitated exactly as described for linear molecule fail to provide a mechanism for the 5-min sample. the synthesis of the 5' ends of the DNA. If RNA is a primer in this system, there would not be a way to fill the gap at the ends upon the removal TABLE 1. Specific activities of the heavy and light strands separated from the HpaI "e" fragment after 5- of RNA, because all the known DNA polymerases require a primer nucleotide sequence beor 10-min labeling fore elongation can occur. This problem has 5 min 10 min been solved by the formation of concatameres Strand for the replication of a molecule such as bacteri"e "c" "e" "C" ophage T7, which replicates as a linear DNA 3.1 0.8 2.2 2.3 Heavy (32). Concatameres, which are joined molecules longer than unit length, allow the end of one 0.5 7.8 0.5 6.5 Light molecule to serve as a primer for another. No a The "e" fragment was obtained from the same such covalently linked concatameres have been DNAs as shown in Fig. 6 and 7. The "e" heavy found during adenovirus DNA replication, alstrand corresponds to the DNA plotted as (0) in Fig. though small quantities of viral DNA may sedi6 and 7, and the specific activities (pulse-labeled ment faster than genome length on alkaline PH]DNA/uniformly labeled [14C]DNA) shown were calculated as for the figures. Data for the "c" frag- gradients (3). These larger molecules have never been shown to be labeled with the kinetment from Fig. 6 and 7 are shown for comparison. ics expected of replicating intermediates, are reported to be linked to host cell DNA, and may not allow the usual types of DNA-DNA interac- be important in the integration of viral DNA in tions at the ends to facilitate circularization of host chromosomes even during the lytic cycle. The data presented in this report are consistduplex DNA. Recently, a protein that holds the ends of adenovirus DNA together has been pur- ent with one of the models for bidirectional ified with the DNA from virions (22). However, replication, originally reported by Sussenbach no circular forms have yet been recognized as et al. (27). However, our observations are not consistent with Sussenbach's proposal that repintermediates in adenovirus DNA synthesis. It is also possible that there is a nonspecific lication on the displaced strand often begins at 5'-exonuclease digesting small portions of the one or several internal initiation sites. AlDNA. If these regions were repaired, thymi- though our data do not specify on which molecdine label would appear at the ends of the com- ular end the first round of displacement synthepleted DNA molecules. Of the three proposed sis occurs, Sussenbach proposed that replicaexplanations, only the model of a replicating tion always began at the right end by displaccircle would also explain the specific activity of ing the heavy parental strand. His observation 2.5 r
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of the displacement of only the heavy strand has been questioned by several other investigators (15, 28). This asymmetrical model of bidirectional replication for adenovirus DNA is similar to models reported for the replication of mitochondrial DNA (21). Another similarity between these two systems is the relative resistance of the replication of both DNAs to inhibitors of protein synthesis (14, 26). The uncoupling of DNA synthesis from its usual strict dependence on new protein synthesis may depend in part on the displacement model ofreplication shared by both mitochondrial and adenovirus DNAs. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant CA-11502 from the National Cancer Institute. Marshall S. Horwitz is the recipient of a Public Health Service Career Development Award from the National Cancer Institute (1K04 CA-35554). I wish to thank Arthur Davino for expert technical assistance, Jerard Hurwitz for helpful discussions, and Stephen Baum, Susan Horwitz, and Matthew Scharff for critical reading of the manuscript. I also wish to thank Julian Pan for providing EcoRI enzyme for some ofthe pilot studies and Martin Farber for furnishing the bacterial strains used to produce the restriction endonucleases. LITERATURE CITED 1. Arrand, J. R., W. Keller, and R. J. Roberts. 1974. Extent of terminal repetition in adenovirus 2 DNA. Cold Spring Harbor Symp. Quant. Biol. 39:401-407. 2. Bellet, A. J. D., and H. B. Younghusband. 1972. Replication of the DNA of chick embryo lethal orphan virus. J. Mol. Biol. 72:691-709. 3. Burger, H., and W. Doerfler. 1975. Intracellular forms of adenovirus DNA. III. Integration of the DNA of adenovirus type 2 into host DNA in productively infected cells. J. Virol. 13:975-992. 4. Danna, K. J., and D. Nathans. 1972. Bidirectional replication of simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 69:309-310. 5. Dintzis, H. 1961. Assembly of peptide chains of hemoglobin. Proc. Natl. Acad. Sci. U.S.A. 57:247-261. 6. Doerfler, W., and A. K. Kleinschmidt. 1970. Denaturation pattern of the DNA of adenovirus type 2 as determined by electron microscopy. J. Mol. Biol. 50:579-593. 7. Ellens, D. J., J. D. Sussenbach, and H. S. Janz. 1974. Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA. Virology 61:427-442. 8. Garon, C. F., K. W. Berry, and J. A. Rose. 1972. A unique form of terminal redundancy in adenovirus DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 69:2391-2395. 9. Green, M. 1962. Studies on the biosynthesis of viral DNA. Cold Spring Harbor Symp. Quant. Biol. 27:219-233. 10. Green, M., M. Pifia, R. Kimes, P. C. Wensink, L. A. MacHattie, and C. A. Thomas, Jr. 1967. Adenovirus DNA. I. Molecular weight and conformation. Proc. Natl. Acad. Sci. U.S.A. 57:1302-1309. 11. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369.
J. VIROL. 12. Horwitz, M. S. 1971. Intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid. J. Virol. 8:675-683. 13. Horwitz, M. S. 1974. Location of the origin of DNA replication in adenovirus type 2. J. Virol. 13:10461054. 14. Horwitz, M. S., C. Brayton, and S. G. Baum. 1973. Synthesis of type 2 adenovirus DNA in the presence of cycloheximide. J. Virol. 11:544-551. 15. Lavelle, G., C. Patch, G. Khoury, and J. Rose. 1975. Isolation and partial characterization of singlestranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA. J. Virol. 16:775-782. 16. Magnusson, G., V. Pigiet, E. L. Winnacker, R. Abrams, and P. Reichard. 1973. RNA-linked DNA fragments during polyoma DNA replication. Proc. Natl. Acad. Sci. U.S.A. 70:412-415. 17. Maizel, J. V., Jr., D. 0. White, and M. D. Scharff. 1968. The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of type 2, 7a and 12. Virology 36:115-125. 18. Mulder, C., J. R. Arrand, H. Delius, W. Keller, U. Pettersson, R. J. Roberts, and P. A. Sharp. 1974. Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases EcoRI and HpaI. Cold Spring Harbor Symp. Quant. Biol. 39:397-400. 19. Mulder, C., and H. Delius. 1972. Specificity of the break produced by restriction endonuclease R1 in simian virus 40 DNA as revealed by partial denaturation mapping. Proc. Natl. Acad. Sci. U.S.A. 69:3215-3219. 20. Okazaki, R. T., T. Okazaki, K. Sakabe, K. Sugimoto, R. Kainung, A. Sugino, and N. Iwatsuki. 1968. In vitro mechanism of DNA chain growth. Cold Spring Harbor Symp. Quant. Biol. 33:129-143. 21. Robberson, D. L., H. Kasamatsu, and J. Vinograd. 1972. Replication of mitochondrial DNA. Circular replicative intermediates in mouse L cells. Proc. Natl. Acad. Sci. U.S.A. 69:737-741. 22. Robinson, A. J., H. B. Younghusband, and A. J. D. Bellett. 1973. A circular DNA-protein complex from adenoviruses. Virology 56:54-69. 23. Schilling, R., B. Weingartner, and E. L. Winnacker. 1975. Adenovirus type 2 DNA replication. II. Termini of DNA replication. J. Virol. 16:767-774. 24. Sharp, P. A., P. H. Gallimore, and S. J. Flint. 1974. Mapping of adenovirus 2 RNA sequences in lyrically infected cells and transformed cell lines. Cold Spring Harbor Symp. Quant. Biol. 39:457474. 25. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose ethidium bromide electrophoresis. Biochemistry
12:3055-3063. 26. Storrie, B., and G. Attardi. 1972. Expression of the mitochondrial genome in HeLa cells. XIII. Effect of selective inhibition of cytoplasmic or mitochondrial protein synthesis on mitochondrial nucleic acid synthesis. J. Mol. Biol. 71:177-199. 27. Susenbach, J. S., P. C. Van der Vliet, D. J. Ellens, and H. S. Janz. 1972. Linear intermediates in the replication of adenovirus DNA. Nature (London) New Biol.
239:4749. 28. Tolun, A., and U. Pettersson. 1975. Termination sites for adenovirus type 2 DNA replication. J. Virol. 16:759-766. 29. Van der Eb, A. J. 1973. Intermediates in type 5 adenovirus DNA replication. Virology 51:11-23. 30. Van der Vliet, P. C., and J. S. Sussenbach. 1972. The mechanism of adenovirus DNA synthesis in isolated nuclei. Eur. J. Biochem. 30:584-592. 31. VlIak, J. M., T. H. Rozin, and J. S. Sussenbach. 1975. Studies on the mechanism of replication of adenovi-
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rus DNA. IV. Discontinuous DNA chain propagation. Virology 63:168-175. 32. Watson, J. D. 1972. Origin of concatemeric T7 DNA. Nature (London) New Biol. 239:197-201. 33. Winnacker, E. L. 1975. Adenovirus type 2 DNA replica-
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tion. I. Evidence for discontinuous DNA synthesis. J. Virol. 15:744-758. 34. Wolfson, J., and D. Dressier. 1972. Adenovirus-2 DNA contains an inverted terminal repetition. Proc. Natl. Acad. Sci. U.S.A. 69:3054-3057.
Vol. 18, No. 1 Printed in U-SA.
Bidirectional Replication of Adenovirus Type 2 DNA MARSHALL S. HORWITZ Department ofMicrobiology-Immunology, Cell Biology, and Pediatrics, Albert Einstein College ofMedicine, Bronx, New York 10461 Received for publication 11 November 1975
After short periods of labeling with [3H]thymidine, recently completed adenovirus DNA molecules were isolated and cleaved with restriction endonucleases. The strands (heavy and light) of most of the restriction endonuclease fragments were separated. The pattern of labeling clearly shows an asymmetry of radioactivity on the isolated strands of each restriction endonuclease piece. The data is consistent with replication proceeding in the 5' to 3' direction on each strand. Thus, there is an initiation point placed at or near each end of the molecule. Adenovirus type 2 DNA, a linear molecule of 23 x 106 daltons, replicates in the nucleus and produces approximately 100,000 copies/cell during lytic infection (9, 10). The duplex DNA has nonpermuted sequences (6) and inverted terminal redundancy at ends (8, 34), which are identical for 100 to 140 nucleotide pairs (1). The inverted terminal redundancy allows the formation of single-strand (ss) circles by base pairing between both ends ofthe denatured ss molecules. Since the ends of adenovirus duplex DNA are identical, these molecules cannot be converted to covalently linked double-strand (ds)
circles like those formed by bacteriophage lambda DNA (10, 34). During replication, ss molecules larger than the genome have not been demonstrated (12, 29, 30); therefore, no covalent addition of progeny DNA to parental molecules occurs. There are small pieces of adenovirus DNA similar to "Okazaki fragments" (20), which can be dissociated from the replication complex by alkaline denaturation (2, 12, 31, 33). During normal viral replication it is not known if all regions of the genome are first polymerized into Okazaki fragments, which are subsequently joined. Replicating molecules have a significantly higher buoyant density than parental viral DNA (27, 29). Further evidence has shown that the density shift is caused by extensive ss DNA regions and not by RNAprimer fragments as reported for polyoma DNA replication (16). Recently, a number of models of replication, which include bidirectional growth, have been proposed for adenovirus DNA. Sussenbach and co-workers have presented data that replication starts at the right end (AT rich) by displacing the parental heavy strand with continuous polymerization in the 5' to 3' direction on the light-strand template (7). After a delay, replication starts on the displaced strand at various internal points or may
start at the 3' end of the template molecule in a pattern of continuous growth in the opposite direction to that on the first strand. Experiments from our laboratory, in which adenovirus DNA half-molecules were produced by mechanical shearing, have shown that there is bidirectional growth with two termination sites, one on the left and another on the right half of the molecule (13). The present study extends these findings by using two restriction endonucleases, which have allowed us to examine nine regions of the DNA. The method to determine the origin and terminus of DNA replication is similar to that employed by Dintzis (5) to analyze the direction of replication of polypeptides and more recently by Danna and Nathans (4) to isolate the origin and direction of replication of simian virus 40 DNA. When a radioactive precursor is added to a system synthesizing DNA, the label enters replicating molecules at a growing point that is at a different site in each molecule. This assumes that the labeling procedure does not change the rate of DNA synthesis by synchronizing molecules at any phase of the replication cycle. The molecules, which are completed during labeling periods shorter than the total synthesis time of the macromolecule, will be preferentially labeled at the terminus. Therefore, when completed molecules are effectively separated from replicating molecules, the amount of radioactivity in various regions of the completed molecules will reflect the origin and direction of synthesis. In this report the pattern of labeling of ds DNA indicates highest specific activity at both ends of the DNA, which is consistent with a termination site at each end of the molecule. While this manuscript was in preparation, similar results were reported (23, 28). However, examination of the single-strands 307
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of each of these DNA fragments has shown that the heavy and light strands label asynchronously. This difference in labeling is most consistent with replication in which each strand is synthesized in the 5' to 3' direction starting at opposite ends of the molecule. (A preliminary report of these results was presented at the Cold Spring Harbor Tumor Virus Meeting, Cold Spring Harbor, N. Y., August, 1975.) MATERIALS AND METHODS Cells and viruses. The source of HeLa cells, adenovirus type 2, and the conditions of infection have been previously described (17). All experiments were done in suspension cultures at an input multiplicity of 4,000 virions/cell (100 to 200 PFU/cell). Radioactive labeling of cells. At 18 h postinfection, the infected HeLa cells were centrifuged for 2 min at 1,500 rpm in an International PRJ centrifuge and resuspended at a concentration of 107 cells/ml in Eagle spinner medium with 5% fetal calf serum. After 5 min of temperature equilibration in a water bath at 37 C, the cells were radioactively labeled with [3H]thymidine at 0.25 mCi/ml (40 to 60 Ci/ mmol). The incorporation of radioactivity was stopped by diluting the cells in 7 volumes of ice-cold Earle salts, rapidly centrifuging the cells at 1,500 rpm, and adding 0.2% sodium dodecyl sulfate in 0.01 M Tris-EDTA (pH 7.4). Using these conditions, the incorporation of radioactivity was linear for 60 min without any appreciable lag at the beginning of this interval (14). Purification of viral DNA. Intact [3H]thymidinelabeled viral DNA was purified from cells after precipitating large-molecular-weight cell DNA by a modification of the Hirt procedure (11, 29, 30). Cells (3 x 107) were suspended in 2 ml of 0.01 M Tris, 0.01 M EDTA (pH 7.4) at 0 C. Sodium dodecyl sulfate (0.2%) and 500 ,ug of Pronase (preincubated at 37 C for 2 h to digest any residual nucleases) were added, and the mixture was incubated for 15 min at 30 C. The volume was increased to 9 ml by the addition of the Tris-EDTA buffer, which contained 1% sodium dodecyl sulfate. After a 5-min incubation at 30 C, NaCl was added to a final concentration of 1 M. The solution was left at 4 C for 16 h, and the precipitate was removed by centrifugation at 12,000 rpm for 20 min in a Spinco angle 30 rotor. By processing 1.5 x 107 to 3 x 107 cells in a final volume of 10 ml, 80% of newly replicated adenovirus DNA was recovered in the Hirt supernatant. Viral DNA was precipitated from the supernatant with 2 volumes of ethanol. The DNA, redissolved in 1 ml of 0.01 x SSC (SSC = 0.15 M NaCl + 0.015 M sodium citrate), was centrifuged in an SW27 rotor of the Spinco ultracentrifuge (16 h at 22,000 rpm) on 16-ml, 5 to 20% neutral sucrose gradients containing 1 M NaCl, 0.01 M phosphate buffer, and 0.01 M EDTA. The 31S fractions were pooled, dialyzed against 0.3 M NaCl, 0.01 M Tris, 0.01 M EDTA (pH 8.1), and loaded onto 2-ml columns of benzoyl-naphthoyl-DEAE-cellulose (BND-
J. VIROL. cellulose). The ds DNA was eluted with 1 M NaCl, and the DNA containing any ss region was eluted with 1 M NaCl and 2% caffeine in the same TrisEDTA buffer (29). The appropriate column fractions were precipitated with 2 volumes of ethanol and redissolved in 0.01 x SSC. ['4C]thymidine-labeled viral DNA, which was used as a uniformly labeled marker, was purified by disrupting virion which had been banded twice on CsCl density gradients (12). Restriction endonucleases. The enzymes from both Escherichia coli (EcoRI) and Haemophilus parainfluenzae (HpaI) were purified from bacterial strains obtained from the Cold Spring Harbor Laboratory. The bacteria were grown and the enzymes were purified as previously described (19, 25). All endonuclease digests were incubated for 4 h at 37 C in 10 mM Tris-hydrochloride (pH 7.4) with 10 mM MgCl2, 6 mM KCl, and 1 mM dithiothreitol. The reaction was stopped in 0.04 M EDTA; the solution was adjusted to a final concentration of 10% sucrose and 0.1% bromophenol blue. The DNA fragments were separated by electrophoresis on cylindrical gels (1.6 by 35 cm) of 1.4% agarose in Tris-EDTA-acetate (TEA = 40 mM Tris-hydrochloride, 1 mM EDTA, 5 mM sodium acetate) buffer for 16 h at 100 V. The gels were stained in the same TEA buffer containing 0.5 ,ug of ethidium bromide per ml, and the bands were visualized with a UV-light source (25). For separation of the strands of each restriction endonuclease fragment of DNA, the ds fragments were cut out of the agarose gel after staining with ethidium bromide and visualization by minimal exposure to UV irradiation. The short cyclindrical piece of gel was placed into a glass scintillation vial with 10 ml of 0.2 M NaOH for 2.5 h at room temperature. The NaOH was decanted, and the gel was soaked for a further 2 h in TEA electrophoresis buffer at 0 C. The gel slice was placed back into the electrophoresis tube, and a new column of gel was polymerized over the original slice. The agarose was poured after equilibration at 60 C. After the polymerization, the gel tube was inverted, and the electrophoresis was performed under identical conditions as used for the original separation of the ds fragments. The gel were stained with ethidium bromide and the single strands were visualized. The separated strands were cut from the gel, and the radioactivity was counted. Radioactivity in the gel slices was quantitated by remelting the agarose in an autoclave and then adding 10 ml of scintillation fluid [1 part Triton, 2 parts toluene, 5 g of 2,5-diphenyloxazole (PPO) per liter, and 50 mg of 1,4-bis-(5-phenyloxazolyl)benzene (POPOP) per liter], which had been heated to 50 C. The samples, which were shaken immediately after the addition of scintillation fluid, were cooled to room temperature and counted in the ambient temperature scintillation counter (Beckman LS 230). Reagents. [3Hlthymidine (40 to 60 Ci/mmol) and ['4C]thymidine (57 mCi/mmol) were purchased from Schwarz BioResearch, Inc. Agarose was obtained from Sargent Welch Co. BND-cellulose was purchased from Serva.
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cules on BND-cellulose, which eliminates any DNA with ss regions. The results of BND-cellulose chromatography of the 5-, 10-, 15-, and 240min samples are shown in Fig. 2. The first peak to elute (fraction 2) contains the completely ds molecules, and the second peak (fraction 8) represents any molecules with ss regions. Greater than 99.3% of DNA, purified from virion and similarly treated, elutes in the ds region. When DNA from the virion is denatured by boiling before chromatography, 100% of the DNA subsequently elutes in fraction 8, the ss region (data not shown). With the shortest pulse times examined (5 min), most of the DNA from the 31S region of the gradient has some ss regions, which are on molecules that have just initiated replication. By 15 min, the 31S region of the gradient has more labeled ds than ss molecules, and by 4 h 76% of the labeled DNA is in ds molecules. Determination of specific activity of ds regions of pulse-labeled DNA. The ds[3H]- thymidine-labeled DNA from fraction 2 (Fig. 2) was mixed with uniformly labeled [14C]thymidinecontaining DNA isolated from the adenovirion. The DNAs were digested either with the restriction endonuclease EcoRI or a mixture of this enzyme and that derived from HpaI. The patterns of digestion for EcoRI and HpaI have ') t0 10 been elucidated (18). The pertinent restriction endonuclease pieces are drawn to scale on the abscissa of Fig. 4. The EcoRI pieces are designated in capital letters (B to F) and the 0. HpaI fragments are designated in lowercase letters (e, c, f, a). "C-" and "E-" refer to EcoRI fragments further digested by HpaI with the loss of approximately 1.5% of the genome. The results of a typical EcoRI + HpaI restriction endonuclease digest of the mixture of [3H]DNA pulse-labeled for 5 min and the [U'4C]viral DNA are shown in Fig. 3. Peaks with the higher ratios of 3H- to '4C-labeled DNA include "c," "C-," and "e," which are near both ends of the molecules (see Fig. 4). A summary I0 top WCTION NUMBER of results obtained from 5-, 10-, and 15-min FIG. 1. Sedimentation velocity gradients to sepa- pulse-labeled DNAs are shown in Fig. 4. The rate replicating from completed viral DNA. Pulse- lowest specific activity (3H/14C) is in the region labeled DNA isolated from the Hirt supernatant was of fractions "a" and "B" near the center of the centrifuged on neutral sucrose gradients as described molecule, and the specific activity increases toin Materials and Methods. The gradients were frac- ward both ends. As expected, differences in the tionated into 0.75-ml aliquots, and the radioactivity specific activity of the individual pieces are was determined by removing 20 pl from each fraction greatest for the 5-min digest, less at 10 min, for quantitating the trichloroacetic acid-precipitable absent by 15 min. DNA. Fractions in the region of 31S-completed viral and minor or of specific activity clearly shows This pattern DNA (fraction 14) were pooled as designated by the bar, and the DNA was dialyzed against 0.3 M NaCl, bidirectional growth of the molecule, but a 0.01 M EDTA, and 0.01 M Tris (pH 8.1) before number of quite different models could explain chromatography on BND-cellulose (Fig. 2). Symbols: this data. 0, 5-min pulse; 0, 15-min pulse. To differentiate between numerous possibiliRESULTS Isolation of recently completed viral DNA molecules. Eighteen hours postinfection, cells were radioactively labeled with [3H]thymidine and viral DNA was separated from host DNA by using the Hirt procedure. The Hirt supernatant, containing replicating and completed viral DNA molecules, was centrifuged on neutral sucrose gradients as shown in Fig. 1. The completed molecules (31S) sediment to the region in the gradient that peaks at fraction 14. The heterogeneous population of replicating molecules sediment faster and appear between fractions 1 and 14 (29). The completed molecules were further purified free of replicating mole-
u0
C-)
C-)
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20
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.10
Ca.2
~~~~~~~~~~~~~~~~~~~~~5
I
5
10
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N U M B E R F R A C T IO N from completed viral DNA. Two-milliliter to replicating separate FIG. 2. BND-cellulose chromatography columns ofBND-cellulose were prepared in 2.5-ml disposable plastic syringes by wetting the cellulose in 0.3 M NaCl, 0.01 M EDTA, 0.01 M Tris buffer (pH 8.1) containing 20% ethanol. The ethanol was removed by centrifugation of the resin immediately after suspension of the cellulose. The BND was poured into the columns and extensively washed with the same buffer without ethanol until the optical density of the effluent at 260 nm was less than 0.05. The DNA, which had been dialyzed against the column buffer, was loaded onto the BND and washed with 12 ml of the same buffer. Elution of ds DNA was achieved with 12 ml of the column buffer to which NaCI had been added to a final concentration of 1 M. Aliquots (2 ml) were collected during the elation, and 50-pJ samples were removed for quantitation of the DNA in each fraction. The column was then washed with 12 ml of the latter buffer, to which 2% caffeine (designated by the arrow) had been added. The ss DNA elated after the addition of caffeine and was quantitated by taking 50-pl aliquots for radioactivity counting. The ds DNA, elated in fraction 2, was precipitated with 2 volumes of ethanol. (A) *, 5 min; 0, 10 min. (B) A, 15 min; A, 240 min.
ties, it was necessary to know if replication was similar on both strands. Sharp et al. (24) have shown that the denatured heavy (H) and light (L) strands of most of the restriction endonuclease fragments can be successfully separated on agarose gels. Using the alkaline denaturation technique for DNA embedded in agarose (see above), we have been able to separate H and L strands from the EcoRI "B." "C," "D," "E," and "F" pieces and the HpaI "e" and "c" pieces. Determination of specific activity of ss regions of pulse-labeled DNA. The ratio of 3H (pulse)- to 14C (uniformly)-labeled DNA was determined for the separated strands of each restriction endonuclease piece isolated from recently completed duplex DNA as described in Materials and Methods. The extent of strand separation for several of the fragments is shown
in Fig. 5. The strand separation of DNA denatured within the agarose gel is superior to denaturation in solution not only because of the relative speed of the former technique but also because of the decreased amount of renatured DNA found after electrophoresis. In most experiments, as in the one shown in Fig. 5, there is no renatured DNA detectable by ethidium bromide staining. The assignment of strand specificity (H or L) for the isolated strands follows the designation of Sharp et al. (24). The faster moving band for the EcoRI "B." "C," "D," "E," and "F" and the slower band for HpaI "e" and "c" (P. A. Sharp, personal communication) belong to the same strand, which is the heavy strand in alkaline CsCl gradients. The HpaI "a" fragment does not separate into H and L strands under any conditions tried thus far.
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go
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F R AC T I 0 N N U M B E R FIG. 3. Agarose gel electrophoresis of a restriction endonuclease digest of the 5-min pulse-labeled DNA. The ds viral DNA from fraction 2 (Fig. 2), which was extracted from cells pulse-labeled for 5 min with [3H]thymidine, was mixed with ["4C]thymidine-containing DNA purified from adenovirions. The mixture was digested with EcoRI and HpaI for 4 h as described in Materials and Methods. After electrophoresis of the DNA on 1.4% agarose columns for 16 h at 100 V, the gel was stained with ethidium bromide and the DNA bands were located. The gel was sliced into aliquots starting with a cut between the 'a" and Sc" pieces to insure that these closely migrating fractions were in separate gel slices. The gels were melted, and the radioactivity was quantitated by scintillation counting. The designation of the restriction endonuclease fragments is as described in the text. The location of the fragments and their relative sizes are shown on the abscissa ofFig. 4. Fragments "E-" and "F' do not separate in this system.
In Fig. 6 and 7, a summary of the specific activities of the isolated strands is shown for pulse-labeling periods of 5 and 10 min, respectively. It is clear that the specific activity is quite different for the corresponding regions on both strands, and this suggests that the L strand (0) is replicating from left to right and the H strand (0) from right to left. Singlestrand data for the "e" fragment at the extreme left end of the molecule is not shown in the figures but is presented in Table 1. The high specific activity on the L strand of the "e" frag-. ment and the low specific activity on the H strand do not continue the trend of labeling derived from quantitating replication on the other 96% of the molecule. DISCUSSION The data is most consistent with bidirectional growth, which is asymmetrical on each
of the strands of adenovirus type 2 DNA. The light strand appears to initiate at or near the left hand end and replicate continuously to the right. This corresponds to growth in the 5' to 3' direction according to the data of Sharp et al., who assigned the 5' end of the heavy strand to the right (24). In contrast, the heavy strand initiates on the right and replicates toward the lefthand end. This model of replication does not require Okazaki fragments to successfully propagate either chain, although several investigators have reported the presence of these intermediates. There appears to be a discrepancy of labeling at the lefthand end of the molecule, which was detected by examining the HpaI "e" piece representing 4% of the DNA. The light "e" strand had more radioactivity than expected for an origin of replication. This could occur because the origin of replication contains nucleotides
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such as RNA serving a primer function. Since licating molecule is a circle, and that replicathe RNA would have to be excised and replaced tion continues beyond the molecular end to an with deoxynucleotides, this region may be la- extent of 4% of the genome. Although linear beled by thymidine with kinetics similar to a adenovirus DNA molecules have terminal reterminus. This would occur if the RNA were dundancy, it is of the inverted type, which does removed toward the end of the replication on that particular strand. 3 OrAnother possible explanation is that the rep1.2*
2
5k
20 0 15
0
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-
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05
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e
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FIG. 6. Order of labeling (5 min) of selected regions of ss adenovirus DNA. DNA was labeled with e c f a B F D E C [3H]thymidine for 5 min in adenovirus-infected cells RESTRICTION ENDONUCLEASE FRAGMENT at 18 h postinfection. The recently completed duplex FIG. 4. Order of labeling of selected regions of ds viral DNA was purified and digested either with adenovirus DNA. From data such as that in Fig. 3, EcoRI for fragments B, F, D, E, and C or with EcoRI ratios ofpulse-labeled [3H]DNA to uniformly labeled + HpaI for fragments e, c, f, and a; the DNA was ['4C]DNA were calculated. Included are the data electrophoresed on agarose gels as described in from the 5-, 10-, and 15-min pulse-labeling periods 3. Selected fragments were processed to separateFig. the obtained either from digests using EcoRI alone (frag- H and L strands according to the description in Fig. ments B, F, D, E, C) or together with the HpaI en- 5 and in Materials and Methods. The specific activity zyme (fragments e, c, f, a). This results from the of the pulse-labeled DNA (PH) in relation to unithree time points are normalized to give identical formly labeled DNA (14C) was determined and plotratios for the "e" fragment. Symbols: 0, 5 min; 0, ted in relation to its map position on the adenovirus 10 min; A, 15 min. chromosome. Symbols: *, L strand; 0, H strand.
t c, r..>
IR
Hpo. e Hpiior FIG. 5. Strand separation of restriction endonuclease fragments. Pulse-labeled DNA ([3H]thymidine) was mixed with uniformly-labeled DNA (['4C]thymidine) and digested either with EcoRI orHpaI. The pieces were separated on agarose gels, stained, and cut from the gel. After soaking the gel slices in 0.2 M NaOH to denature the DNA, the endonuclease fragments were rerun on neutral agarose gels as described in Materials and Methods. The results of strand separation are shown for several of the fragments. Although the HpaI "a" piece does not separate into H and L strands, the HpaI "c" and the EcoRI "B" fragment separate into two bands. (For other fragments successfully separated by this technique, see text).
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the "e" heavy strand, which is lower than expected for its position next to the "c" fragment. Similar data is not yet available from the qS righthand end of the molecule. The HpaI "g" 20h fragment, which is 1.4% ofthe genome, is at the right end. Although the "g" band was visualized with ethidium bromide staining and apu peared in fraction 44 (Fig. 3), the "g" radioactivity was always superimposed on a background of counts in fractions 45 through 47, which 10 makes exact calculations difficult. We have not yet been able to separate the strands of the "g" fragment and are approaching the quantitation of counts on each of the strands by hybridiza05K tion of duplex "g" fragment with isolated strands of the EcoRI "C" fragment. It is also possible that labeling at the molecu1 IT lar ends may be complicated by an inability to f B F D E C c e separate newly initiated molecules with very RESTRICTION ENDONUCLEASE FRAGMENT FIG. 7. Order of labeling (10 min) of selected re- short replicating regions from the pool of ds, gions of ss adenovirus DNA. Infected cells were la- 31S completed molecules. It is difficult to debeled with [3H]thymidine for 10 min in the same sign a control experiment for this possibility. experiment as described in Fig. 6. The DNA was The models of adenovirus replication as a processed and quantitated exactly as described for linear molecule fail to provide a mechanism for the 5-min sample. the synthesis of the 5' ends of the DNA. If RNA is a primer in this system, there would not be a way to fill the gap at the ends upon the removal TABLE 1. Specific activities of the heavy and light strands separated from the HpaI "e" fragment after 5- of RNA, because all the known DNA polymerases require a primer nucleotide sequence beor 10-min labeling fore elongation can occur. This problem has 5 min 10 min been solved by the formation of concatameres Strand for the replication of a molecule such as bacteri"e "c" "e" "C" ophage T7, which replicates as a linear DNA 3.1 0.8 2.2 2.3 Heavy (32). Concatameres, which are joined molecules longer than unit length, allow the end of one 0.5 7.8 0.5 6.5 Light molecule to serve as a primer for another. No a The "e" fragment was obtained from the same such covalently linked concatameres have been DNAs as shown in Fig. 6 and 7. The "e" heavy found during adenovirus DNA replication, alstrand corresponds to the DNA plotted as (0) in Fig. though small quantities of viral DNA may sedi6 and 7, and the specific activities (pulse-labeled ment faster than genome length on alkaline PH]DNA/uniformly labeled [14C]DNA) shown were calculated as for the figures. Data for the "c" frag- gradients (3). These larger molecules have never been shown to be labeled with the kinetment from Fig. 6 and 7 are shown for comparison. ics expected of replicating intermediates, are reported to be linked to host cell DNA, and may not allow the usual types of DNA-DNA interac- be important in the integration of viral DNA in tions at the ends to facilitate circularization of host chromosomes even during the lytic cycle. The data presented in this report are consistduplex DNA. Recently, a protein that holds the ends of adenovirus DNA together has been pur- ent with one of the models for bidirectional ified with the DNA from virions (22). However, replication, originally reported by Sussenbach no circular forms have yet been recognized as et al. (27). However, our observations are not consistent with Sussenbach's proposal that repintermediates in adenovirus DNA synthesis. It is also possible that there is a nonspecific lication on the displaced strand often begins at 5'-exonuclease digesting small portions of the one or several internal initiation sites. AlDNA. If these regions were repaired, thymi- though our data do not specify on which molecdine label would appear at the ends of the com- ular end the first round of displacement synthepleted DNA molecules. Of the three proposed sis occurs, Sussenbach proposed that replicaexplanations, only the model of a replicating tion always began at the right end by displaccircle would also explain the specific activity of ing the heavy parental strand. His observation 2.5 r
I
0
or
If
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of the displacement of only the heavy strand has been questioned by several other investigators (15, 28). This asymmetrical model of bidirectional replication for adenovirus DNA is similar to models reported for the replication of mitochondrial DNA (21). Another similarity between these two systems is the relative resistance of the replication of both DNAs to inhibitors of protein synthesis (14, 26). The uncoupling of DNA synthesis from its usual strict dependence on new protein synthesis may depend in part on the displacement model ofreplication shared by both mitochondrial and adenovirus DNAs. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant CA-11502 from the National Cancer Institute. Marshall S. Horwitz is the recipient of a Public Health Service Career Development Award from the National Cancer Institute (1K04 CA-35554). I wish to thank Arthur Davino for expert technical assistance, Jerard Hurwitz for helpful discussions, and Stephen Baum, Susan Horwitz, and Matthew Scharff for critical reading of the manuscript. I also wish to thank Julian Pan for providing EcoRI enzyme for some ofthe pilot studies and Martin Farber for furnishing the bacterial strains used to produce the restriction endonucleases. LITERATURE CITED 1. Arrand, J. R., W. Keller, and R. J. Roberts. 1974. Extent of terminal repetition in adenovirus 2 DNA. Cold Spring Harbor Symp. Quant. Biol. 39:401-407. 2. Bellet, A. J. D., and H. B. Younghusband. 1972. Replication of the DNA of chick embryo lethal orphan virus. J. Mol. Biol. 72:691-709. 3. Burger, H., and W. Doerfler. 1975. Intracellular forms of adenovirus DNA. III. Integration of the DNA of adenovirus type 2 into host DNA in productively infected cells. J. Virol. 13:975-992. 4. Danna, K. J., and D. Nathans. 1972. Bidirectional replication of simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 69:309-310. 5. Dintzis, H. 1961. Assembly of peptide chains of hemoglobin. Proc. Natl. Acad. Sci. U.S.A. 57:247-261. 6. Doerfler, W., and A. K. Kleinschmidt. 1970. Denaturation pattern of the DNA of adenovirus type 2 as determined by electron microscopy. J. Mol. Biol. 50:579-593. 7. Ellens, D. J., J. D. Sussenbach, and H. S. Janz. 1974. Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA. Virology 61:427-442. 8. Garon, C. F., K. W. Berry, and J. A. Rose. 1972. A unique form of terminal redundancy in adenovirus DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 69:2391-2395. 9. Green, M. 1962. Studies on the biosynthesis of viral DNA. Cold Spring Harbor Symp. Quant. Biol. 27:219-233. 10. Green, M., M. Pifia, R. Kimes, P. C. Wensink, L. A. MacHattie, and C. A. Thomas, Jr. 1967. Adenovirus DNA. I. Molecular weight and conformation. Proc. Natl. Acad. Sci. U.S.A. 57:1302-1309. 11. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369.
J. VIROL. 12. Horwitz, M. S. 1971. Intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid. J. Virol. 8:675-683. 13. Horwitz, M. S. 1974. Location of the origin of DNA replication in adenovirus type 2. J. Virol. 13:10461054. 14. Horwitz, M. S., C. Brayton, and S. G. Baum. 1973. Synthesis of type 2 adenovirus DNA in the presence of cycloheximide. J. Virol. 11:544-551. 15. Lavelle, G., C. Patch, G. Khoury, and J. Rose. 1975. Isolation and partial characterization of singlestranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA. J. Virol. 16:775-782. 16. Magnusson, G., V. Pigiet, E. L. Winnacker, R. Abrams, and P. Reichard. 1973. RNA-linked DNA fragments during polyoma DNA replication. Proc. Natl. Acad. Sci. U.S.A. 70:412-415. 17. Maizel, J. V., Jr., D. 0. White, and M. D. Scharff. 1968. The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of type 2, 7a and 12. Virology 36:115-125. 18. Mulder, C., J. R. Arrand, H. Delius, W. Keller, U. Pettersson, R. J. Roberts, and P. A. Sharp. 1974. Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases EcoRI and HpaI. Cold Spring Harbor Symp. Quant. Biol. 39:397-400. 19. Mulder, C., and H. Delius. 1972. Specificity of the break produced by restriction endonuclease R1 in simian virus 40 DNA as revealed by partial denaturation mapping. Proc. Natl. Acad. Sci. U.S.A. 69:3215-3219. 20. Okazaki, R. T., T. Okazaki, K. Sakabe, K. Sugimoto, R. Kainung, A. Sugino, and N. Iwatsuki. 1968. In vitro mechanism of DNA chain growth. Cold Spring Harbor Symp. Quant. Biol. 33:129-143. 21. Robberson, D. L., H. Kasamatsu, and J. Vinograd. 1972. Replication of mitochondrial DNA. Circular replicative intermediates in mouse L cells. Proc. Natl. Acad. Sci. U.S.A. 69:737-741. 22. Robinson, A. J., H. B. Younghusband, and A. J. D. Bellett. 1973. A circular DNA-protein complex from adenoviruses. Virology 56:54-69. 23. Schilling, R., B. Weingartner, and E. L. Winnacker. 1975. Adenovirus type 2 DNA replication. II. Termini of DNA replication. J. Virol. 16:767-774. 24. Sharp, P. A., P. H. Gallimore, and S. J. Flint. 1974. Mapping of adenovirus 2 RNA sequences in lyrically infected cells and transformed cell lines. Cold Spring Harbor Symp. Quant. Biol. 39:457474. 25. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose ethidium bromide electrophoresis. Biochemistry
12:3055-3063. 26. Storrie, B., and G. Attardi. 1972. Expression of the mitochondrial genome in HeLa cells. XIII. Effect of selective inhibition of cytoplasmic or mitochondrial protein synthesis on mitochondrial nucleic acid synthesis. J. Mol. Biol. 71:177-199. 27. Susenbach, J. S., P. C. Van der Vliet, D. J. Ellens, and H. S. Janz. 1972. Linear intermediates in the replication of adenovirus DNA. Nature (London) New Biol.
239:4749. 28. Tolun, A., and U. Pettersson. 1975. Termination sites for adenovirus type 2 DNA replication. J. Virol. 16:759-766. 29. Van der Eb, A. J. 1973. Intermediates in type 5 adenovirus DNA replication. Virology 51:11-23. 30. Van der Vliet, P. C., and J. S. Sussenbach. 1972. The mechanism of adenovirus DNA synthesis in isolated nuclei. Eur. J. Biochem. 30:584-592. 31. VlIak, J. M., T. H. Rozin, and J. S. Sussenbach. 1975. Studies on the mechanism of replication of adenovi-
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rus DNA. IV. Discontinuous DNA chain propagation. Virology 63:168-175. 32. Watson, J. D. 1972. Origin of concatemeric T7 DNA. Nature (London) New Biol. 239:197-201. 33. Winnacker, E. L. 1975. Adenovirus type 2 DNA replica-
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