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Initiation of adenovirus type 2 DNA replication
VIROLOGY
72, 89-98 (1976)
initiation
of Adenovirus
PIERRE
Type 2 DNA Replication’
BOURGAUX,2 LOUIS DELBECCHI, BOURGAUX-RAMOISY
AND DANIELLE DPpartement
de Microbiologic,
Centre
Hospitalier Universitaire, Universite’ Qubbec, Canada JlH 5N4 Accepted
February
de Sherbrooke,
Sherbrooke,
25,1976
Fourteen to eighteen hours after low multiplicity infection of monolayer cultures of KB cells with adenovirus type 2 (Ad. 2), viral DNA was labeled with short pulses of tritiated thymidine and selectively extracted using sodium deoxycholate. After it had been purified, viral DNA was fractionated into partly single-stranded (or replicating) molecules and double-stranded (or completed) molecules on columns of benzolyatednaphthoylated DEAE (BND) cellulose. Completed molecules were cleaved with endonuclease R, from E. coli prior to electrophoresis through agarose-acrylamide slab gels. In agreement with previous results (Winnacker, 1974; Schilling et al ., 1975), tritium recovery in the resulting fragments indicated that termination of replication occurred at the molecular ends of the genome. Replicating molecules were exposed to a single-strand specific nuclease from Neurospora crassa before being treated with endonuclease R,. In this instance, maximal tritium recovery was obvserved for the R, fragments corresponding to the center of the adenovirus DNA molecule. These results suggest that replication originates at the center of the molecule and proceeds in the direction of both ends.
tars, replicating adenovirus DNA contains extended single-stranded regions which complicate its analysis by restriction endonucleases. As pointed out by Tolun and Pettersson (19751, replicating adenovirus DNA with such properties has been obtained under extraordinary experimental conditions, such as infection at high multiplicities or synthesis in isolated nuclei. We have already described some properties of the replicating viral DNA which we isolated from monolayers of KB cells infected with adenovirus type 2 (Ad. 2) at low multiplicity (Robin et al., 1973); Bourgaux-Ramoisy et al., 1974). Under our conditions, replicating adenovirus DNA was found to contain relatively little material which could be digested by single-strand specific nucleases (Robin et al., 1973). It appeared under the electron microscope as Y-shaped molecules of genome length, with no visible single-stranded regions, while no partly single-stranded, unbranched, molecules could be observed
INTRODUCTION
Restriction endonuclease have been used successfully to ascertain the origin, terminus, and direction of replication in DNA from SV40 (Danna and Nathans, 1972; Nathans and Danna, 1972; Fareed et al., 1972) and polyoma virus (Crawford et al., 1974). In these experiments, conclusions were drawn from the analysis of both the replicating and the completed molecules, labeled after short pulses of tritiated thymidine . The study of the mode of replication of adenovirus DNA was recently approached in a similar manner. Thus far, experiments have been performed on completed molecules exclusively (Winnacker, 1974; Schilling et al., 1975; Tolun and Pettersson, 1975). Presumably, this is due to the fact that, in the hands of most investiga’ A preliminary account of this work was presented at the Third International Congress for Virology, Madrid, September 1975. * Author to whom reprint requests should be addressed. 89 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
90
BOURGAUX,
DELBECCHI
(Bourgaux-Ramoisy et al., 1974). Quite clearly, such material was likely to provide a suitable substrate for restriction enzymes. The endonuclease R, from E. coli has been shown to cleave Ad. 2 DNA into six unique fragments (Pettersson et al., 1973). We have used it to analyze both completed and replicating molecules of Ad. 2 DNA, labeled after short pulses. As described below, the results we obtained with the completed molecules are in complete agreement with those already published by others. Together with the data on the distribution of label in replicating molecules, they clearly invalidate some of the models already proposed for adenovirus DNA replication. MATERIALS
AND
METHODS
Virus. The origin of the Ad. 2 we used, the conditions for its growth in monolayers of KB cells, and most of the techniques employed here have already been described (Bourgaux-Ramoisy et al., 1974; Bourgaux and Bourgaux-Ramoisy, 1971; Bourgaux et al., 1971). Isolation
of pulse-labeled
viral
DNA.
Confluent monolayer cultures of KB cells (approx 2 x lo6 cells per petri dish) were exposed to Ad. 2 (2 x lo6 to 4 x lo6 PFU in 0.2 ml) for 90 min at 37”. They were then covered with Dulbecco’s modified Eagle’s medium containing 2.5% calf serum and further incubated for 12 to 16 hr. In order to label DNA, medium was discarded and each dish received 0.75 ml of fresh medium containing 15 pg/ml of &fluorodeoxyuridine (Bourgaux-Ramoisy et al., 1974), followed 15 min later by an additional 0.25 ml of the same medium containing 50 PCi of [3H]thymidine (New England Nuclear, sp act 20 Ci/mmol). After a 3- to 9-min pulselabeling period, the cultures were cooled on crushed ice, washed with buffered saline solution and covered with a 1% (w/v) sodium deoxycholate solution (in 0.02 M EDTA, 0.02 M Tris-HCI buffer, pH 8.6). The resulting lysate was scraped from the plates, clarified by centrifugation, and treated for 4 hr at 37” with sodium sarcosinate and pronase, as already described (Bourgaux et al., 1971; Bourgaux-Ramoisy et al., 1974).
AND
BOURGAUX-RAMOISY
After bringing the density to 1.45 g/ml using solid Cs2S04, the DNA solution was centrifuged for 18 hr at 50,000 rpm in the A321 rotor of a B60 International ultracentrifuge. Fractions were collected from the bottom of the tubes and those containing acid-precipitable tritium activity, representing about 0.5 ml, were mixed and layered on top of 4 ml of a CsCl solution (in 0.02 M EDTA, 0.02 M Tris-HCl, pH 8.6) of density @) 1.715 g/ml. The latter was centrifuged in the A321 rotor at 50,000 rpm for 42 hr prior to fraction collection. After determination of the radioactivity in the fractions, those containing the labeled viral DNA (1.725 g/ml < p < 1.710 g/ml) were pooled and stored at -20”. BND-cellulose chromatography. The pool from the CsCl gradient containing the viral DNA was added to 1 vol of water and 2 vol of 0.3 M NaCl, 0.001 M EDTA, 0.01 M Tris-HCl buffer, pH 8.1. The mixture was loaded on a column of benzoylated-naphthoylated DEAE-cellulose (Serva, Heidelberg). Batch elution of the DNA was performed as already described (Bourgaux et al., 1971; Bourgaux-Ramoisy et al., 1974). Mature DNA was eluted with 0.65 M NaCl and replicating DNA with 1 M NaCl plus 2% caffeine. Marker DNAs. We have described previously the preparation of 32P-labeled DNA from adenovirus (Bourgaux-Ramoisy et al., 1974). Intracellular viral DNA was obtained from KB cells which had been incubated in the presence of [32P]orthophosphate (Amersham, 50 @XI ml) from the sixth to the eighteenth hr after infection. It was extracted, purified, and fractionated on BND-cellulose as described above for DNA pulse-labeled with r3H]thymidine. Marker SV40 DNA labeled with 14C was obtained from CV-1 cells by a procedure similar to that described for polyoma DNA (Bourgaux and BourgauxRamoisy, 1972), except that labeling was continuous from the eighteenth to the fortieth hour after infection, and that viral DNA was extracted by the Hirt (1967) method. Enzymes. Both endonuclease R, and endonuclease from Neurospora crassa were obtained from Miles. Endonuclease RI digestion was performed in 0.01 MgCl, and
REPLICATION
OF
0.09 M Tris-HCl, pH 8.0, as described by Pettersson et al. (1973). N. CMSSU digestion was carried out as described previously (Robin et al., 19731, except that 0.09 M Tris-HCl, pH 8.0 was used instead of 0.01 M Tris-HCl, pH 7.5. Agarose-acrylamide
gel electrophoresis.
Immediately after R, digestion was completed, samples were layered on top of composite slab gels cast in a #220 Hoefer apparatus. The gels contained 0.7% agarose (Seakem, Bausch and Lomb) and 2.2% recrystallized acrylamide (Eastman Kodak). Electrophoresis was carried out at 30 V for 18 hr in a buffer containing 0.0025 M EDTA, 0.09 M Tris, 0.09 M Boric acid (pH 8.4). The gels were dried and subjected to autoradiography using RP-14 Kodak film. After superimposing the gel over the autoradiogram, the portions of gel containing 32P were delineated with a pencil, cut out with fine scissors, and incubated at 37” for 18 hr in scintillation vials containing 0.2 ml of hydrogen peroxide. To each vial was added 3 ml of Triton X-100 and 2.4 ml of PPO-POPOP-toluene solution, and the samples were counted in a scintillation spectrometer. Some of the autoradiograms were scanned with a Corning 750 microdensitometer, equipped with an integrator. RESULTS
Buoyant Density DNA
of Replicating
Ad.
2
In earlier experiments, we found little evidence of extended single-stranded regions in replicating molecules of Ad. 2 DNA, as judged from either appearance in the electron microscope (Bourgaux-Ramoisy et al., 1974) or susceptibility to singlestrand specific nucleases (Robin et al., 1973). We. also failed to notice buoyant density differences between virion DNA and pulse.labeled viral DNA, or purified replicating viral DNA, under our standard conditions for banding DNA in CsCl solutions (unpublished results). Since these findings were in apparent conflict with published results (Sussenbach et al., 1972; Bellett and Younghusband, 1972; van der Eb, 1973; Pettersson, 1973), we decided to reinvestigate the density of replicating DNA, using high resolution density gra-
ADENOVIRUS
DNA
91
dients. Replicating Ad. 2 DNA, labeled and purified on BND-cellulose (see Materials and Methods), was mixed with two suitable markers and centrifuged to equilibrium in CsCl solution, using an angle rotor. A density difference of 3 mg/ml, too small to have been noticed previously, could then be detected between replicating and mature Ad. 2 DNA (Fig. la). This difference could be abolished (data not shown) by treating the replicating DNA with single-strand specific nucleases from either Neurospora crassa or Aspergillus oryzae (SJ, as already described (Robin et al., 1973). Taking into consideration that the maximal increment in density one could expect from single-strandedness is 16 mg/ml (Vinograd et al., 1963), the observed increment may indicate that singlestranded regions represent one-fifth of the length of the replicating molecules. This estimate of the proportion of singlestranded material in replicating Ad. 2 DNA is in agreement with previous calculations (Robin et al., 1973). We speculated that the relative paucity of single-stranded material that we have consistently observed (Robin et al., 1973; Bourgaux-Ramoisy et al., 1974; this work, Fig. 1) in replicating adenovirus DNA was possibly due to the experimental conditions selected. With the hope of identifying the source of the disagreement, we performed a number of experiments in which we altered some of our conditions. More specifically, we used multiplicities ranging from 1 to 100 PFU per cell during virus infection, the Hirt (1967) procedure instead of our regular method (see Materials and Methods) for viral DNA isolation, and phenol extraction rather than pronase treatment, as a first step in DNA purification. An average density difference of 3 to 4 mg/ml was consistently observed between virion DNA and replicating DNA under all these circumstances, except one: In three experiments where various multiplicities of infection were used, an increment in density of 6 to 7 mg/ml was observed for the replicating DNA obtained at a multiplicity of 100 PFU, i.e., 3000 physical particles, per cell (Fig. lb). As already observed by Pettersson (19731, suppression of such a difference required the use,
BOURGAUX,
DELBECCHI
AND BOURGAUX-RAMOISY
of replicating DNA with the multiplicity of infection. In any event, we felt our experimental conditions were advantageous in at least one respect. Consisting largely in doublestranded DNA, the replicating molecules we had isolated were likely to represent a suitable substrate for restriction endonucleases.
a
too
Analysis of Completed Molecules Labeled after Short Pulses -1
-(
00 E 6 I Y ? k
x00
100
fraction
no
FIG. 1. Buoyant density of replicating Ad. 2 DNA. Eighteen hours after infection with Ad. 2, KB cells were pulse-labeled for 10 min with 13Hlthymidine, as described in Materials and Methods. Viral DNA was selectively extracted, purified after equilibrium density gradient centrifugation in Cs$O4 and CsCl solutions, and fractionated by BND-cellulose chromatography. The DNA recovered with caffeine solution was mixed with 32P-labeled mature Ad. 2 DNA @ = 1.716 g/ml) and W-labeled SV40 DNA form I @ = 1.699 g/ml) and centrifuged for 72 hr in CsCl solution (p = 1.715 g/ml), using the angle rotor A321 (Materials and Methods). The distribution of radioactivity through the gradients was determined at the end of the run. Some cells had been infected at a multiplicity of 1 PFUlcell (a), others at a multiplicity of 100 PFU/cell (b). In (b), note the pronounced asymmetry of the 3H peak.
not only of a single-strand specific nuclease, but also of pancreatic ribonuclease (not shown). At present, we have no explanation for these changes in properties
KB cells, infected under our standard conditions, were pulse-labeled with 13Hlthymidine for 3, 6, or 9 min as described in Materials and Methods. After extraction, purification and BND-cellulose fractionation of the viral DNA, the material recovered in the saline fraction was concentrated by alcohol precipitation and redissolved in the buffer used for nuclease digestion. Using 32P-labeled viral DNA as a marker, it was first verified that all of this material comigrated with DNA extracted from virions during both sedimentation through neutral sucrose gradients and electrophoresis through agarose-acrylamide gels (not shown). Such 13Hlthymidine-labeled completed molecules were then mixed with the same 32P-labeled marker and treated with endonuclease R,. The resulting fragments were separated by electrophoresis, recovered from the dried gels after autoradiography (Fig. 2), and analyzed in a scintillation spectrometer set for the counting of 32P and ?I% Irrespective of the length of the pulse, the 3H to 32P ratio was found to be low for fragments B and F, which originate from the center of the Ad. 2 molecule, and for the very large fragment A (Fig. 3). For the shortest pulse, fragment F was clearly the least labeled, while two gradients of labeling diverging from that fragment in the direction of both ends were discernible. Closely similar results have already been published by other workers (Winnacker, 1974; Schilling et al., 1975; Tolun and Pettersson, 1975), who have concluded that termini of replication exists at both molecular ends of Ad. 2 DNA.
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OF ADENOVIRUS
93
DNA
Accordingly, the caffeine-eluted replicating molecules collected in the experiment described in the preceding section, were treated with N. crassa nuclease and fractionated by velocity sedimentation through neutral sucrose gradients (Fig. 4). As observed earlier (Robin et al., 19731, a high proportion (46% in the experiment illustrated) of the radioactive material generated by the nuclease was recovered as pieces cosedimenting with marker Ad. 2 fragment
order
A
B FD
6t
E C
.
5’ FIG. 2. Separation on agarose-acrylamide slab gel of R, fragments from Y2P-labeled mature Ad. 2 DNA extracted from cells. Uniformly labeled completed molecules, purified by BND-cellulose chromatography, were treated with endonuclease R,. The resulting fragments were separated by electrophoresis as described in Materials and Methods. After autoradiography for 40 hr (bottom), slices containing the 3zP-labeled fragments were cut out from the gel and counted in a scintillation spectrometer. Bands A, B, C, D, E, and F (visible from left to right on the autoradiogram) were found to represent, respectively, 877 (58.9%), 173 (11.6%1, 168 (11.3%), 114 (7.7%), 89 (6.0%), and 67 (4.5%) cpm. A microdensitometer tracing of the autoradiogram is also shown (top). Integration of the area under the peaks indicated that fragments A to F accounted for 59.0, 12.5, 11.5, 6.6, 5.5, and 4.5% .of the total absorbance. These figures are closely similar to those obtained by Pettersson et al. (1973) for DNA extracted from Ad. 2 virus.
Analysis of Replicating after Short Pulses
DNA
Labeled
In a previous report (Robin et al., 19731, we have described how replicating Ad. 2 DNA can be cleaved into double-stranded “pieces” of high molecular weight, using a single-strand specific nuclease from Neurospora crassa. We speculated that such pieces could provide us with direct information on the location of the origin of replication.
0
025
0.50 fractional
0.75
1
length
FIG. 3. Relative yield of tritium of R, fragments from completed molecules labeled with 13H]thymidine. Viral DNA, which had been pulselabeled for 3 (01, 6 (A), and 9 min CO), was fractionated by BND-cellulose chromatography. Completed molecules, recovered from the column with saline solution, were mixed with Ad. 2 DNA uniformly labeled with 32P and treated with endonuclease R,. After gel electrophoresis and autoradiography, slices containing the 32P-labeled bands were cut out from the gel and counted. The figure shows the 3H/ 3zP ratios for the bands corresponding to the various fragments, ordered as shown at the top (Mulder et al., 1974). Corrections were made for differences in thymidine content of individual fragments, estimated from the 3H/32P ratios of fragments from DNA uniformly labeled with 132Plorthophosphate and 13Hlthymidine. As also done in Fig. 5, ratios in the figure are normalized to fragment F. Normalizing to fragment A, for which absolute tritium yield was highest, produced similar curves. The data for 3 and 9 min were obtained in the same experiment; those for 6 min are average values from two additional experiments.
94
BOLJRGAUX, 1000
a
DELBECCHI 500
500 L
- 250
400 -
- 250
FIG. 4. Sedimentation properties of intact and nuclease-treated replicating Ad. 2 DNA. Viral DNA, labeled after a 6-min pulse with 13Hlthymidine was subjected to BND-cellulose fractionation. Replicating DNA was eluted from the column with a caffeine solution. A small aliquot was kept untreated and the remainder was treated with the nuclease from Neurospora cmssa (see Materials and Methods). Both the untreated and the nuclease-treated DNA were mixed with 32P-labeled mature Ad. 2 DNA and centrifuged through 5 to 20% neutral sucrose gradients for 72 min at 60,000 rpm in the SB405 rotor. After gradient fractionation, radioactivity was determined on either the whole of the fractions (untreated DNA, a) or an aliquot (nuclease-treated DNA, b). In the latter case, pools comprising respectively large (L), medium size (Ml, and small ‘pieces” (S) were made from successive fractions. From Studier’s equation (19651, we calculated that the lower limit for L pieces was 15 x lo6 daltons and the upper limit for S pieces, 5 x lo6 daltons. For comparison, fragment A and B cleaved from Ad. 2 DNA by endonuclease R, have molecular weights of 13.6 x lo6 and 2.7 x 106, respectively (Pettersson et al.. 1973).
DNA. Assuming that, under our experimental conditions, replicating DNA would consist primarily of Y-shaped molecules, such pieces could only represent structures
AND BOURGAUX-RAMOISY
made of the unreplicated portion and of one of the growing branches of the replicating molecules (Robin et al., 1973). The sedimentation profile also indicated the presence of smaller fragments. However, no fragments of molecular weight lower than 2 x lo6 (13.5 S; Studier, 1965) can be detected after N. CMSS~ digestion. This suggests that replicating molecules contain few, rather than multiple, singlestranded regions. We pooled separately the fractions from the gradients comprising the large (L), medium size (Ml, and small (S) pieces, and then proceeded with the analysis by R, digestion as already described for the completed molecules. The striking feature of the results was that, in pieces of all size classes and for all labeling times, the highest relative tritium yield was generally observed in fragment F (Fig. 5). A second maximum was observed (for fragemnt E) in the M and S pieces: Since total tritium recovery from the gels was not excellent in those cases (see Fig. 51, too much emphasis should not be put on the last finding however. Low tritium yield in fragment A was to be expected in classes M and S, since this fragment did equal, or exceed, in size the pieces in those classes (see Fig. 4). As L pieces had mostly the same molecular weight as the completed molecules, the results registered for these pieces appear particularly meaningful. In these pieces, the pattern of labeling actually appears as the mirror image of that of the completed molecules (see Fig. 3). The 3H/ 32P ratio is highest for F, and decreases in the direction of both molecular ends. The decline in the 3H/32P ratio is sharper towards the right of the molecule. It is also more pronounced with longer labeling times. These results are what one would expect if the large pieces were derived from molecules replicating bidirectionally from a centrally located origin (Nathans and Danna, 1972; Danna and Nathans, 1972). Analysis of Uniformly ing Molecules
Labeled Replicat-
If replication were to proceed from the center of the molecule in the two opposite
REPLICATION
OF ADENOVIRUS fragment
, B ,F.D,E.C
A
A
95
DNA
order , B ,F,D,E,C
A
, B .F.D.E,C
I
I
a2 -
0
0.25
0.50
0.75
1-o
0.50 0.75 025 fractional length
1-o
0.25
0.50
0.75
1
FIG. 5. Relative yield of R, fragments from replicating molecules labeled with 13Hlthymidine. The replicating DNA which, in the experiments referred to in Fig. 3, had been recovered from the BND-cellulose columns with caffeine solution, was treated withN. crossa nuclease and fractionated as shown in Fig. 4. The large (L), medium size (M), and small “pieces” (S) were mixed with Ad. 2 DNA uniformly labeled with 3zP and treated with endonuclease R,. After electrophoresis and autoradiography, the portions of the gel comprising the six 3ZP-labeled bands were cut out and counted for both 3H and 3zP. The 3H/32P ratios are corrected for the thymidine content of each fragment and normalized to F. Pulse-labeling with 13Hlthymidine had been for 3 (O), 6 (A), and 9 min (0). In each panel, the curves appear rather similar, although they show different slopes. This indicates that both N. crossa cleavage and sucrose gradient fractionation worked consistently for different DNA preparations, or even in separate experiments. In the case of the medium size and small “pieces” (M and S), total tritium recoveries however represented only 80 and 508, respectively, of what they were for large pieces or completed molecules. Presumably, this was due to the existence of 3H-labeled fragments of intermediate size which did not corn&-ate, and thus were not counted, with any of the 32P-labeled fragments (see also Fig. 6).
directions, digestion of replicating molecules by endonuclease R, should produce more of the fragments corresponding to the center than of the fragments at either end. No such over-representation of the central portion of the mature molecule would be expected for replicating DNA in the hypothesis of displacement synthesis, proceeding from either one (Ellens et al., 1974) or both molecular ends @chilling et al., 1975). We thus performed the following experiment. KB cells were pulse-labeled with 32P-orthophosphate from the sixth to the eighteenth hr after infection. Viral DNA was then extracted, purified, and fractionated as described in Materials and Methods. Replicating molecules were treated with endonuclease R, prior to elec-
trophoresis through composite gels. The yields of 3*P of bands A to F were measured and compared (Fig. 6) to those of the same bands generated from mature DNA in the same experiment (see Fig. 2). Quantitation of the radioactive material in the six bands, performed by two different methods, was complicated by the presence of fragments of intermediate size in the R, digest from the replicating molecules. Consistent results were nevertheless obtained. The comparison with mature DNA suggested that fragment F is the most frequently represented in partially duplicated molecules. In contrast, fragments A, C, D, E, and, among these, terminal fragments A and C, all appear under-represented in comparison to the centrally lo-
96
BOURGAUX,
DELBECCHJ
AND BOURGAUX-RAMOISY
formed using 32P-labeled replicating DNA digested by both endonuclease R, and the nuclease from N. crussa. Similar results were obtained (not shown). DISCUSSION
FIG. 6. R, fragments from replicating molecules uniformly labeled with 32P. Replicating DNA, eluted with caffeine solution from a BND-cellulose column, was treated with endonuclease R, prior to electrophoresis through an agarose-acrylamide gel. After autoradiography for 48 hr, slices corresponding to the six main radioactive bands (bottom) and to the gel between these bands were cut out and counted. The optical density of the autoradiogram was also monitored (top). Bands A, B, C, D, E, and F represented 66% of the total radioactivity thus recovered from the gel. When the radioactivity of the individual bands was related to that of the same bands from mature DNA (see Fig. 2), the following ratios were obtained after normalizing to F: A, 0.49; B, 0.92; F, 1; D, 0.87; E, 0.82; C, 0.78. When calculated from the optical density scans, these ratios were: A, 0.61; B, 0.80; F, 1; D, 0.83; E, 0.77; C, 0.75.
cated fragments B and F. The markedly lower yield of fragment A presumably results from both its terminal location and its large size. The latter property makes it more likely for the corresponding stretch of DNA to be only partially duplicated in the replicating molecule, and thus to migrate anomalously in the gel after R, cleavage. It should be pointed out, however, that the representation of the five other fragments is not related to size, as fragment B is more abundant than the smaller fragments C, D, and E. Finally, the same experiment was per-
The observations we made using endonuclease R1 to cleave completed molecules labeled after short pulse of 13Hlthymidine are in perfect agreement with those already reported by others (Winnacker, 1974; Schilling et al., 1975; Tolun and Pettersson, 1975). These observations thus confirm that termini for replication exist at or near both molecular ends of Ad. 2 DNA. They also suggest that temination and, by inference, initiation of replication proceeds under our conditions as already observed by others, even though some of the properties of the replicating molcules are seemingly unique to our system. The existence of termination points for replication at both molecular ends can be reconciled with all models envisaged thus far for adenovirus DNA replication, except one, namely that assuming the synchronous synthesis of the two complementary chains from a single origin located at a unique end of the molecule. Our analysis of the uniformly labeled replicating molecules is also consistent with termini existing at both ends. After R1 digestion of such molecules, low relative yields of fragments A and C were obtained. Fragments B, F, and D were found at a higher frequency, as it would be expected if, during replication, the central portion of the molecule was completed first. Aside from these quantitative differences, and from the occurrence of fragments of intermediate size in the digest from replicating molecules, the latter molecules and the mature ones produced remarkably similar electrophoresis patterns after R, digestion (Figs. 2 and 6). This is not too surprising, for the nuclease from N. CF~SS~ generates from replicating molecules a very substantial amount of material with the sedimentation properties of mature molecules. After exposure of uniformly labeled replicating DNA to the single-strand specific nuclease, as much as 50% of the radioactive material recovered
REPLICATION
OF
was found in such large “pieces” (Robin et al., 1973). Assuming replication of Ad. 2 DNA would operate as postulated by Sussenbach et al., (1972) for Ad. 5 DNA, pieces of the size of the whole genome could only originate from branched molecules, as a result of digestion, or cleavage, of the displaced strand by the single-strand specific nuclease. Such pieces should include the origin of replication, which in Sussenbath’s model (Ellens et al., 1974), is located at the right molecular end of the viral DNA. Yet, after short pulses of [3H]thymidine the relative yields of tritium in fragments D, E, and C were always low. Moreover, the relative yields of these three fragments of comparable size always constituted a very steep gradient in the order D > E > C, as it would be expected if the origin, or one of the origins, of replication was located at the left of D. Therefore, we feel our data clearly exclude displacement synthesis from a unique origin located at the right molecular end of the Ad. 2 genome. It would appear that our data also provide rather compelling evidence of a central location of the origin of replication in Ad. 2 DNA, a possibility that has already been envisaged by Winnacker (1974). After short pulse-labeling periods, the central fragments are the least labeled in mature molecules, while being the most labeled in pieces of all sizes derived from replicating molecules (Fig. 5). In replicating molecules, they are also the most frequently represented of all fragments. Such a central location for the origin of replication, however, cannot be reconciled easily with the electron microscopic observations made in several laboratories, including ours, on replicating adenovirus DNA. Specifically, it is difficult to see how a centrally initiated replication process would generate branched intermediates which all appear Y-shaped in the electron microscope (Sussenbach et al., 1972; Ellens et al., 1974; Bourgaux-Ramoisy et al., 1974). Yet, failure to observe the characteristic “eye-shaped” structures in preparations of replicating adenovirus DNA can hardly be regarded as strong evidence against the possibility of replication being actually in-
ADENOVIRUS
DNA
97
itiated from the center of the molecule. In this respect, it should be recalled that the concept of adenovirus DNA replication proceeding by displacement synthesis from a unique end of the molecule was based largely on electron microscopic observations (Sussenbach et al., 1972; Ellens et al., 1974). This concept was recently challenged by several independent groups on the basis of data obtained by restriction enzyme analysis and/or molecular hybridization of pulse-labeled viral DNA (Horwitz, 1974; Winnacker, 1974, Schilling et al., 1975; Tolun and Pettersson, 1975; Lavelle et al., 1975; this work). In the face of direct disagreement between electron microscopic observations and biochemical data, such as those presented herein suggesting a centrally-initiated process, we would therefore tend to favor the latter. At present, we are carrying out experiments with the aim of distinguishing between two alternate modes of replication, that directly supported by the data reported here, and another one, also implying termination at both molecular ends, namely initiation at both ends (Horwitz, 1974; Schilling et al., 1975). ACKNOWLEDGMENTS We thank Dr. E. Bradley for critical reading of the manuscript, and Miss Diane Bourbeau for technical assistance. This work was supported by grants from the Medical Research Council of Canada and from the National Cancer Institute of Canada. REFERENCES BELLETT, A. J. D., and YOUNGHU~BAND, H. B. (1972). Replication of the DNA of chick embryo lethal orphan virus. J. Mol. Biol. 72, 691-709. BOURGAUX, P., and BOURGAIJX-RAMOISY, D. (1971). A symmetrical model for polyoma virus DNA replication. J. Mol. Bid. 62, 513-524. BOURGAUX, P., and BOURGAUX-RAMOIEY, D. (1972). Unwinding of replicating polyoma virus DNA. J. Mol. Biol. 70, 399-413. BOURGAUX, P., BOURGAUX-RAMOISY, D., and SEILER, P. (1971). The replication of the ringshaped DNA of polyoma virus. II. Identification of molecules at various stages of replication. J. Mol. Biol. 59, 195-206. BOURGAUX-RAMOISY, D., ROBIN, J., and BOURGAUX, P. (1974). Replicating DNA of adenovirus type 2. Canad. J. Biochen. 52, 181-189. CRAWFORD, L. V., ROBBINS, A. K., NICXLIN, P. M., and OSBORN, K. (1974). Polyoma DNA replication:
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Location of the origin in different virus strains. Cold Spring Harbor Symp. Quant. Biol. 39, 219225. DANNA, K., and NATHANS, D. (1972). Bidirectional replication of simian virus 40 DNA. Proc. Nat. Acad. Sci. USA 69, 3097-3100. ELLENB, D. J., SUSSENBACH, J. S., and JANSZ, H. S. (19741 Studies on the mechanism of replication of adenovirus DNA. II. Electron microscopy of replicating DNA. Virology 61, 427-442. FAREED, G. C., GAEON, C. F., and SALZMAN, N. P. (19721. Origin and direction of simian virus 40 deoxyribonucleic acid replication. J. Virol. 10, 484-491. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cultures. J. Mol. Biol. 26, 365-369. HORWITZ, M. S. (1974). Location of the origin of DNA replication in adenovirus type 2. J. Virol. 13, 1046-1054. LAVELLE, G., PATCH, C., KHOURY, G., and ROSE, J. (1975). Isolation and partial characterization of single-stranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA. J. Viral. 16, 775-782. MULDER, C., ARRAND, J., DELIUS, H., KELLER, W., PETTER~SON, U., ROBERTS, R. J., and SHAIW, P. (1974). Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases EcoRI and Hpa I. Cold Spring Harbor Symp. Quunt. Biol. 39, 397-400. NATHANS, D., and DANNA, K. J. (1972). Specific origin in SV40 DNA replication. Nature New Biol.
AND
BOURGAUX-RAMOISY
236, 200-202. PJYrTEzaSON, U. (1973). Some unusual properties of replicating adenovirus type 2 DNA. J. Mol. Biol. 81, 521-527. PE~TERSBON, U., MULDER, C., DELIUS, H., and SHARP, P. A. (1973). Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R.R1. PFOC.Nat. Acad. Sci. USA 70, 200-204. ROBIN, J., BOURGAUX-RAMOISY, D., and BOURGAUX, P. (1973). Single-stranded regions in replicating DNA of adenovirus type 2. J. Gen. Virol. 20, 233237. SCHILLING, R., WEING~RTNER, B., WINNACKER, E.L. (1975). Adenovirus type 2 DNA replication. II. Termini of DNA replication. J. Virol. 16,767-774. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373-390. SU~ENBACH, J. S., VAN DER VLIET, P. C., ELLENS, D. J., and JANSZ, H. S. (1972). Linear intermediates in the replication of adenovirus DNA. Nature New Biol. 239, 47-49. TOLUN, A., and PETTERBBON,U. (1975). Termination sites for adenovirus type 2 DNA replication. J. Viral. 16, 759-766. VAN DER EB, A. J. (1973). Intermediates in type 5 adenovirus DNA replication. Virology 51, 11-23. VINOG~AD, J., MORRIS, J., DAVIDSON, N., and DOVE, W. F., JR. (1963). The buoyant behavior of viral and bacterial DNA in alkaline CsCl. Proc. Nat. Acad. Sci. USA 49, 12-17. WINNACHER, E. L. (1974). Origins and termini of adenovirus type 2 DNA replication. Cold Spring Harbor Symp. Quant. Biol. 39, 547-550.
72, 89-98 (1976)
initiation
of Adenovirus
PIERRE
Type 2 DNA Replication’
BOURGAUX,2 LOUIS DELBECCHI, BOURGAUX-RAMOISY
AND DANIELLE DPpartement
de Microbiologic,
Centre
Hospitalier Universitaire, Universite’ Qubbec, Canada JlH 5N4 Accepted
February
de Sherbrooke,
Sherbrooke,
25,1976
Fourteen to eighteen hours after low multiplicity infection of monolayer cultures of KB cells with adenovirus type 2 (Ad. 2), viral DNA was labeled with short pulses of tritiated thymidine and selectively extracted using sodium deoxycholate. After it had been purified, viral DNA was fractionated into partly single-stranded (or replicating) molecules and double-stranded (or completed) molecules on columns of benzolyatednaphthoylated DEAE (BND) cellulose. Completed molecules were cleaved with endonuclease R, from E. coli prior to electrophoresis through agarose-acrylamide slab gels. In agreement with previous results (Winnacker, 1974; Schilling et al ., 1975), tritium recovery in the resulting fragments indicated that termination of replication occurred at the molecular ends of the genome. Replicating molecules were exposed to a single-strand specific nuclease from Neurospora crassa before being treated with endonuclease R,. In this instance, maximal tritium recovery was obvserved for the R, fragments corresponding to the center of the adenovirus DNA molecule. These results suggest that replication originates at the center of the molecule and proceeds in the direction of both ends.
tars, replicating adenovirus DNA contains extended single-stranded regions which complicate its analysis by restriction endonucleases. As pointed out by Tolun and Pettersson (19751, replicating adenovirus DNA with such properties has been obtained under extraordinary experimental conditions, such as infection at high multiplicities or synthesis in isolated nuclei. We have already described some properties of the replicating viral DNA which we isolated from monolayers of KB cells infected with adenovirus type 2 (Ad. 2) at low multiplicity (Robin et al., 1973); Bourgaux-Ramoisy et al., 1974). Under our conditions, replicating adenovirus DNA was found to contain relatively little material which could be digested by single-strand specific nucleases (Robin et al., 1973). It appeared under the electron microscope as Y-shaped molecules of genome length, with no visible single-stranded regions, while no partly single-stranded, unbranched, molecules could be observed
INTRODUCTION
Restriction endonuclease have been used successfully to ascertain the origin, terminus, and direction of replication in DNA from SV40 (Danna and Nathans, 1972; Nathans and Danna, 1972; Fareed et al., 1972) and polyoma virus (Crawford et al., 1974). In these experiments, conclusions were drawn from the analysis of both the replicating and the completed molecules, labeled after short pulses of tritiated thymidine . The study of the mode of replication of adenovirus DNA was recently approached in a similar manner. Thus far, experiments have been performed on completed molecules exclusively (Winnacker, 1974; Schilling et al., 1975; Tolun and Pettersson, 1975). Presumably, this is due to the fact that, in the hands of most investiga’ A preliminary account of this work was presented at the Third International Congress for Virology, Madrid, September 1975. * Author to whom reprint requests should be addressed. 89 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
90
BOURGAUX,
DELBECCHI
(Bourgaux-Ramoisy et al., 1974). Quite clearly, such material was likely to provide a suitable substrate for restriction enzymes. The endonuclease R, from E. coli has been shown to cleave Ad. 2 DNA into six unique fragments (Pettersson et al., 1973). We have used it to analyze both completed and replicating molecules of Ad. 2 DNA, labeled after short pulses. As described below, the results we obtained with the completed molecules are in complete agreement with those already published by others. Together with the data on the distribution of label in replicating molecules, they clearly invalidate some of the models already proposed for adenovirus DNA replication. MATERIALS
AND
METHODS
Virus. The origin of the Ad. 2 we used, the conditions for its growth in monolayers of KB cells, and most of the techniques employed here have already been described (Bourgaux-Ramoisy et al., 1974; Bourgaux and Bourgaux-Ramoisy, 1971; Bourgaux et al., 1971). Isolation
of pulse-labeled
viral
DNA.
Confluent monolayer cultures of KB cells (approx 2 x lo6 cells per petri dish) were exposed to Ad. 2 (2 x lo6 to 4 x lo6 PFU in 0.2 ml) for 90 min at 37”. They were then covered with Dulbecco’s modified Eagle’s medium containing 2.5% calf serum and further incubated for 12 to 16 hr. In order to label DNA, medium was discarded and each dish received 0.75 ml of fresh medium containing 15 pg/ml of &fluorodeoxyuridine (Bourgaux-Ramoisy et al., 1974), followed 15 min later by an additional 0.25 ml of the same medium containing 50 PCi of [3H]thymidine (New England Nuclear, sp act 20 Ci/mmol). After a 3- to 9-min pulselabeling period, the cultures were cooled on crushed ice, washed with buffered saline solution and covered with a 1% (w/v) sodium deoxycholate solution (in 0.02 M EDTA, 0.02 M Tris-HCI buffer, pH 8.6). The resulting lysate was scraped from the plates, clarified by centrifugation, and treated for 4 hr at 37” with sodium sarcosinate and pronase, as already described (Bourgaux et al., 1971; Bourgaux-Ramoisy et al., 1974).
AND
BOURGAUX-RAMOISY
After bringing the density to 1.45 g/ml using solid Cs2S04, the DNA solution was centrifuged for 18 hr at 50,000 rpm in the A321 rotor of a B60 International ultracentrifuge. Fractions were collected from the bottom of the tubes and those containing acid-precipitable tritium activity, representing about 0.5 ml, were mixed and layered on top of 4 ml of a CsCl solution (in 0.02 M EDTA, 0.02 M Tris-HCl, pH 8.6) of density @) 1.715 g/ml. The latter was centrifuged in the A321 rotor at 50,000 rpm for 42 hr prior to fraction collection. After determination of the radioactivity in the fractions, those containing the labeled viral DNA (1.725 g/ml < p < 1.710 g/ml) were pooled and stored at -20”. BND-cellulose chromatography. The pool from the CsCl gradient containing the viral DNA was added to 1 vol of water and 2 vol of 0.3 M NaCl, 0.001 M EDTA, 0.01 M Tris-HCl buffer, pH 8.1. The mixture was loaded on a column of benzoylated-naphthoylated DEAE-cellulose (Serva, Heidelberg). Batch elution of the DNA was performed as already described (Bourgaux et al., 1971; Bourgaux-Ramoisy et al., 1974). Mature DNA was eluted with 0.65 M NaCl and replicating DNA with 1 M NaCl plus 2% caffeine. Marker DNAs. We have described previously the preparation of 32P-labeled DNA from adenovirus (Bourgaux-Ramoisy et al., 1974). Intracellular viral DNA was obtained from KB cells which had been incubated in the presence of [32P]orthophosphate (Amersham, 50 @XI ml) from the sixth to the eighteenth hr after infection. It was extracted, purified, and fractionated on BND-cellulose as described above for DNA pulse-labeled with r3H]thymidine. Marker SV40 DNA labeled with 14C was obtained from CV-1 cells by a procedure similar to that described for polyoma DNA (Bourgaux and BourgauxRamoisy, 1972), except that labeling was continuous from the eighteenth to the fortieth hour after infection, and that viral DNA was extracted by the Hirt (1967) method. Enzymes. Both endonuclease R, and endonuclease from Neurospora crassa were obtained from Miles. Endonuclease RI digestion was performed in 0.01 MgCl, and
REPLICATION
OF
0.09 M Tris-HCl, pH 8.0, as described by Pettersson et al. (1973). N. CMSSU digestion was carried out as described previously (Robin et al., 19731, except that 0.09 M Tris-HCl, pH 8.0 was used instead of 0.01 M Tris-HCl, pH 7.5. Agarose-acrylamide
gel electrophoresis.
Immediately after R, digestion was completed, samples were layered on top of composite slab gels cast in a #220 Hoefer apparatus. The gels contained 0.7% agarose (Seakem, Bausch and Lomb) and 2.2% recrystallized acrylamide (Eastman Kodak). Electrophoresis was carried out at 30 V for 18 hr in a buffer containing 0.0025 M EDTA, 0.09 M Tris, 0.09 M Boric acid (pH 8.4). The gels were dried and subjected to autoradiography using RP-14 Kodak film. After superimposing the gel over the autoradiogram, the portions of gel containing 32P were delineated with a pencil, cut out with fine scissors, and incubated at 37” for 18 hr in scintillation vials containing 0.2 ml of hydrogen peroxide. To each vial was added 3 ml of Triton X-100 and 2.4 ml of PPO-POPOP-toluene solution, and the samples were counted in a scintillation spectrometer. Some of the autoradiograms were scanned with a Corning 750 microdensitometer, equipped with an integrator. RESULTS
Buoyant Density DNA
of Replicating
Ad.
2
In earlier experiments, we found little evidence of extended single-stranded regions in replicating molecules of Ad. 2 DNA, as judged from either appearance in the electron microscope (Bourgaux-Ramoisy et al., 1974) or susceptibility to singlestrand specific nucleases (Robin et al., 1973). We. also failed to notice buoyant density differences between virion DNA and pulse.labeled viral DNA, or purified replicating viral DNA, under our standard conditions for banding DNA in CsCl solutions (unpublished results). Since these findings were in apparent conflict with published results (Sussenbach et al., 1972; Bellett and Younghusband, 1972; van der Eb, 1973; Pettersson, 1973), we decided to reinvestigate the density of replicating DNA, using high resolution density gra-
ADENOVIRUS
DNA
91
dients. Replicating Ad. 2 DNA, labeled and purified on BND-cellulose (see Materials and Methods), was mixed with two suitable markers and centrifuged to equilibrium in CsCl solution, using an angle rotor. A density difference of 3 mg/ml, too small to have been noticed previously, could then be detected between replicating and mature Ad. 2 DNA (Fig. la). This difference could be abolished (data not shown) by treating the replicating DNA with single-strand specific nucleases from either Neurospora crassa or Aspergillus oryzae (SJ, as already described (Robin et al., 1973). Taking into consideration that the maximal increment in density one could expect from single-strandedness is 16 mg/ml (Vinograd et al., 1963), the observed increment may indicate that singlestranded regions represent one-fifth of the length of the replicating molecules. This estimate of the proportion of singlestranded material in replicating Ad. 2 DNA is in agreement with previous calculations (Robin et al., 1973). We speculated that the relative paucity of single-stranded material that we have consistently observed (Robin et al., 1973; Bourgaux-Ramoisy et al., 1974; this work, Fig. 1) in replicating adenovirus DNA was possibly due to the experimental conditions selected. With the hope of identifying the source of the disagreement, we performed a number of experiments in which we altered some of our conditions. More specifically, we used multiplicities ranging from 1 to 100 PFU per cell during virus infection, the Hirt (1967) procedure instead of our regular method (see Materials and Methods) for viral DNA isolation, and phenol extraction rather than pronase treatment, as a first step in DNA purification. An average density difference of 3 to 4 mg/ml was consistently observed between virion DNA and replicating DNA under all these circumstances, except one: In three experiments where various multiplicities of infection were used, an increment in density of 6 to 7 mg/ml was observed for the replicating DNA obtained at a multiplicity of 100 PFU, i.e., 3000 physical particles, per cell (Fig. lb). As already observed by Pettersson (19731, suppression of such a difference required the use,
BOURGAUX,
DELBECCHI
AND BOURGAUX-RAMOISY
of replicating DNA with the multiplicity of infection. In any event, we felt our experimental conditions were advantageous in at least one respect. Consisting largely in doublestranded DNA, the replicating molecules we had isolated were likely to represent a suitable substrate for restriction endonucleases.
a
too
Analysis of Completed Molecules Labeled after Short Pulses -1
-(
00 E 6 I Y ? k
x00
100
fraction
no
FIG. 1. Buoyant density of replicating Ad. 2 DNA. Eighteen hours after infection with Ad. 2, KB cells were pulse-labeled for 10 min with 13Hlthymidine, as described in Materials and Methods. Viral DNA was selectively extracted, purified after equilibrium density gradient centrifugation in Cs$O4 and CsCl solutions, and fractionated by BND-cellulose chromatography. The DNA recovered with caffeine solution was mixed with 32P-labeled mature Ad. 2 DNA @ = 1.716 g/ml) and W-labeled SV40 DNA form I @ = 1.699 g/ml) and centrifuged for 72 hr in CsCl solution (p = 1.715 g/ml), using the angle rotor A321 (Materials and Methods). The distribution of radioactivity through the gradients was determined at the end of the run. Some cells had been infected at a multiplicity of 1 PFUlcell (a), others at a multiplicity of 100 PFU/cell (b). In (b), note the pronounced asymmetry of the 3H peak.
not only of a single-strand specific nuclease, but also of pancreatic ribonuclease (not shown). At present, we have no explanation for these changes in properties
KB cells, infected under our standard conditions, were pulse-labeled with 13Hlthymidine for 3, 6, or 9 min as described in Materials and Methods. After extraction, purification and BND-cellulose fractionation of the viral DNA, the material recovered in the saline fraction was concentrated by alcohol precipitation and redissolved in the buffer used for nuclease digestion. Using 32P-labeled viral DNA as a marker, it was first verified that all of this material comigrated with DNA extracted from virions during both sedimentation through neutral sucrose gradients and electrophoresis through agarose-acrylamide gels (not shown). Such 13Hlthymidine-labeled completed molecules were then mixed with the same 32P-labeled marker and treated with endonuclease R,. The resulting fragments were separated by electrophoresis, recovered from the dried gels after autoradiography (Fig. 2), and analyzed in a scintillation spectrometer set for the counting of 32P and ?I% Irrespective of the length of the pulse, the 3H to 32P ratio was found to be low for fragments B and F, which originate from the center of the Ad. 2 molecule, and for the very large fragment A (Fig. 3). For the shortest pulse, fragment F was clearly the least labeled, while two gradients of labeling diverging from that fragment in the direction of both ends were discernible. Closely similar results have already been published by other workers (Winnacker, 1974; Schilling et al., 1975; Tolun and Pettersson, 1975), who have concluded that termini of replication exists at both molecular ends of Ad. 2 DNA.
REPLICATION
OF ADENOVIRUS
93
DNA
Accordingly, the caffeine-eluted replicating molecules collected in the experiment described in the preceding section, were treated with N. crassa nuclease and fractionated by velocity sedimentation through neutral sucrose gradients (Fig. 4). As observed earlier (Robin et al., 19731, a high proportion (46% in the experiment illustrated) of the radioactive material generated by the nuclease was recovered as pieces cosedimenting with marker Ad. 2 fragment
order
A
B FD
6t
E C
.
5’ FIG. 2. Separation on agarose-acrylamide slab gel of R, fragments from Y2P-labeled mature Ad. 2 DNA extracted from cells. Uniformly labeled completed molecules, purified by BND-cellulose chromatography, were treated with endonuclease R,. The resulting fragments were separated by electrophoresis as described in Materials and Methods. After autoradiography for 40 hr (bottom), slices containing the 3zP-labeled fragments were cut out from the gel and counted in a scintillation spectrometer. Bands A, B, C, D, E, and F (visible from left to right on the autoradiogram) were found to represent, respectively, 877 (58.9%), 173 (11.6%1, 168 (11.3%), 114 (7.7%), 89 (6.0%), and 67 (4.5%) cpm. A microdensitometer tracing of the autoradiogram is also shown (top). Integration of the area under the peaks indicated that fragments A to F accounted for 59.0, 12.5, 11.5, 6.6, 5.5, and 4.5% .of the total absorbance. These figures are closely similar to those obtained by Pettersson et al. (1973) for DNA extracted from Ad. 2 virus.
Analysis of Replicating after Short Pulses
DNA
Labeled
In a previous report (Robin et al., 19731, we have described how replicating Ad. 2 DNA can be cleaved into double-stranded “pieces” of high molecular weight, using a single-strand specific nuclease from Neurospora crassa. We speculated that such pieces could provide us with direct information on the location of the origin of replication.
0
025
0.50 fractional
0.75
1
length
FIG. 3. Relative yield of tritium of R, fragments from completed molecules labeled with 13H]thymidine. Viral DNA, which had been pulselabeled for 3 (01, 6 (A), and 9 min CO), was fractionated by BND-cellulose chromatography. Completed molecules, recovered from the column with saline solution, were mixed with Ad. 2 DNA uniformly labeled with 32P and treated with endonuclease R,. After gel electrophoresis and autoradiography, slices containing the 32P-labeled bands were cut out from the gel and counted. The figure shows the 3H/ 3zP ratios for the bands corresponding to the various fragments, ordered as shown at the top (Mulder et al., 1974). Corrections were made for differences in thymidine content of individual fragments, estimated from the 3H/32P ratios of fragments from DNA uniformly labeled with 132Plorthophosphate and 13Hlthymidine. As also done in Fig. 5, ratios in the figure are normalized to fragment F. Normalizing to fragment A, for which absolute tritium yield was highest, produced similar curves. The data for 3 and 9 min were obtained in the same experiment; those for 6 min are average values from two additional experiments.
94
BOLJRGAUX, 1000
a
DELBECCHI 500
500 L
- 250
400 -
- 250
FIG. 4. Sedimentation properties of intact and nuclease-treated replicating Ad. 2 DNA. Viral DNA, labeled after a 6-min pulse with 13Hlthymidine was subjected to BND-cellulose fractionation. Replicating DNA was eluted from the column with a caffeine solution. A small aliquot was kept untreated and the remainder was treated with the nuclease from Neurospora cmssa (see Materials and Methods). Both the untreated and the nuclease-treated DNA were mixed with 32P-labeled mature Ad. 2 DNA and centrifuged through 5 to 20% neutral sucrose gradients for 72 min at 60,000 rpm in the SB405 rotor. After gradient fractionation, radioactivity was determined on either the whole of the fractions (untreated DNA, a) or an aliquot (nuclease-treated DNA, b). In the latter case, pools comprising respectively large (L), medium size (Ml, and small ‘pieces” (S) were made from successive fractions. From Studier’s equation (19651, we calculated that the lower limit for L pieces was 15 x lo6 daltons and the upper limit for S pieces, 5 x lo6 daltons. For comparison, fragment A and B cleaved from Ad. 2 DNA by endonuclease R, have molecular weights of 13.6 x lo6 and 2.7 x 106, respectively (Pettersson et al.. 1973).
DNA. Assuming that, under our experimental conditions, replicating DNA would consist primarily of Y-shaped molecules, such pieces could only represent structures
AND BOURGAUX-RAMOISY
made of the unreplicated portion and of one of the growing branches of the replicating molecules (Robin et al., 1973). The sedimentation profile also indicated the presence of smaller fragments. However, no fragments of molecular weight lower than 2 x lo6 (13.5 S; Studier, 1965) can be detected after N. CMSS~ digestion. This suggests that replicating molecules contain few, rather than multiple, singlestranded regions. We pooled separately the fractions from the gradients comprising the large (L), medium size (Ml, and small (S) pieces, and then proceeded with the analysis by R, digestion as already described for the completed molecules. The striking feature of the results was that, in pieces of all size classes and for all labeling times, the highest relative tritium yield was generally observed in fragment F (Fig. 5). A second maximum was observed (for fragemnt E) in the M and S pieces: Since total tritium recovery from the gels was not excellent in those cases (see Fig. 51, too much emphasis should not be put on the last finding however. Low tritium yield in fragment A was to be expected in classes M and S, since this fragment did equal, or exceed, in size the pieces in those classes (see Fig. 4). As L pieces had mostly the same molecular weight as the completed molecules, the results registered for these pieces appear particularly meaningful. In these pieces, the pattern of labeling actually appears as the mirror image of that of the completed molecules (see Fig. 3). The 3H/ 32P ratio is highest for F, and decreases in the direction of both molecular ends. The decline in the 3H/32P ratio is sharper towards the right of the molecule. It is also more pronounced with longer labeling times. These results are what one would expect if the large pieces were derived from molecules replicating bidirectionally from a centrally located origin (Nathans and Danna, 1972; Danna and Nathans, 1972). Analysis of Uniformly ing Molecules
Labeled Replicat-
If replication were to proceed from the center of the molecule in the two opposite
REPLICATION
OF ADENOVIRUS fragment
, B ,F.D,E.C
A
A
95
DNA
order , B ,F,D,E,C
A
, B .F.D.E,C
I
I
a2 -
0
0.25
0.50
0.75
1-o
0.50 0.75 025 fractional length
1-o
0.25
0.50
0.75
1
FIG. 5. Relative yield of R, fragments from replicating molecules labeled with 13Hlthymidine. The replicating DNA which, in the experiments referred to in Fig. 3, had been recovered from the BND-cellulose columns with caffeine solution, was treated withN. crossa nuclease and fractionated as shown in Fig. 4. The large (L), medium size (M), and small “pieces” (S) were mixed with Ad. 2 DNA uniformly labeled with 3zP and treated with endonuclease R,. After electrophoresis and autoradiography, the portions of the gel comprising the six 3ZP-labeled bands were cut out and counted for both 3H and 3zP. The 3H/32P ratios are corrected for the thymidine content of each fragment and normalized to F. Pulse-labeling with 13Hlthymidine had been for 3 (O), 6 (A), and 9 min (0). In each panel, the curves appear rather similar, although they show different slopes. This indicates that both N. crossa cleavage and sucrose gradient fractionation worked consistently for different DNA preparations, or even in separate experiments. In the case of the medium size and small “pieces” (M and S), total tritium recoveries however represented only 80 and 508, respectively, of what they were for large pieces or completed molecules. Presumably, this was due to the existence of 3H-labeled fragments of intermediate size which did not corn&-ate, and thus were not counted, with any of the 32P-labeled fragments (see also Fig. 6).
directions, digestion of replicating molecules by endonuclease R, should produce more of the fragments corresponding to the center than of the fragments at either end. No such over-representation of the central portion of the mature molecule would be expected for replicating DNA in the hypothesis of displacement synthesis, proceeding from either one (Ellens et al., 1974) or both molecular ends @chilling et al., 1975). We thus performed the following experiment. KB cells were pulse-labeled with 32P-orthophosphate from the sixth to the eighteenth hr after infection. Viral DNA was then extracted, purified, and fractionated as described in Materials and Methods. Replicating molecules were treated with endonuclease R, prior to elec-
trophoresis through composite gels. The yields of 3*P of bands A to F were measured and compared (Fig. 6) to those of the same bands generated from mature DNA in the same experiment (see Fig. 2). Quantitation of the radioactive material in the six bands, performed by two different methods, was complicated by the presence of fragments of intermediate size in the R, digest from the replicating molecules. Consistent results were nevertheless obtained. The comparison with mature DNA suggested that fragment F is the most frequently represented in partially duplicated molecules. In contrast, fragments A, C, D, E, and, among these, terminal fragments A and C, all appear under-represented in comparison to the centrally lo-
96
BOURGAUX,
DELBECCHJ
AND BOURGAUX-RAMOISY
formed using 32P-labeled replicating DNA digested by both endonuclease R, and the nuclease from N. crussa. Similar results were obtained (not shown). DISCUSSION
FIG. 6. R, fragments from replicating molecules uniformly labeled with 32P. Replicating DNA, eluted with caffeine solution from a BND-cellulose column, was treated with endonuclease R, prior to electrophoresis through an agarose-acrylamide gel. After autoradiography for 48 hr, slices corresponding to the six main radioactive bands (bottom) and to the gel between these bands were cut out and counted. The optical density of the autoradiogram was also monitored (top). Bands A, B, C, D, E, and F represented 66% of the total radioactivity thus recovered from the gel. When the radioactivity of the individual bands was related to that of the same bands from mature DNA (see Fig. 2), the following ratios were obtained after normalizing to F: A, 0.49; B, 0.92; F, 1; D, 0.87; E, 0.82; C, 0.78. When calculated from the optical density scans, these ratios were: A, 0.61; B, 0.80; F, 1; D, 0.83; E, 0.77; C, 0.75.
cated fragments B and F. The markedly lower yield of fragment A presumably results from both its terminal location and its large size. The latter property makes it more likely for the corresponding stretch of DNA to be only partially duplicated in the replicating molecule, and thus to migrate anomalously in the gel after R, cleavage. It should be pointed out, however, that the representation of the five other fragments is not related to size, as fragment B is more abundant than the smaller fragments C, D, and E. Finally, the same experiment was per-
The observations we made using endonuclease R1 to cleave completed molecules labeled after short pulse of 13Hlthymidine are in perfect agreement with those already reported by others (Winnacker, 1974; Schilling et al., 1975; Tolun and Pettersson, 1975). These observations thus confirm that termini for replication exist at or near both molecular ends of Ad. 2 DNA. They also suggest that temination and, by inference, initiation of replication proceeds under our conditions as already observed by others, even though some of the properties of the replicating molcules are seemingly unique to our system. The existence of termination points for replication at both molecular ends can be reconciled with all models envisaged thus far for adenovirus DNA replication, except one, namely that assuming the synchronous synthesis of the two complementary chains from a single origin located at a unique end of the molecule. Our analysis of the uniformly labeled replicating molecules is also consistent with termini existing at both ends. After R1 digestion of such molecules, low relative yields of fragments A and C were obtained. Fragments B, F, and D were found at a higher frequency, as it would be expected if, during replication, the central portion of the molecule was completed first. Aside from these quantitative differences, and from the occurrence of fragments of intermediate size in the digest from replicating molecules, the latter molecules and the mature ones produced remarkably similar electrophoresis patterns after R, digestion (Figs. 2 and 6). This is not too surprising, for the nuclease from N. CF~SS~ generates from replicating molecules a very substantial amount of material with the sedimentation properties of mature molecules. After exposure of uniformly labeled replicating DNA to the single-strand specific nuclease, as much as 50% of the radioactive material recovered
REPLICATION
OF
was found in such large “pieces” (Robin et al., 1973). Assuming replication of Ad. 2 DNA would operate as postulated by Sussenbach et al., (1972) for Ad. 5 DNA, pieces of the size of the whole genome could only originate from branched molecules, as a result of digestion, or cleavage, of the displaced strand by the single-strand specific nuclease. Such pieces should include the origin of replication, which in Sussenbath’s model (Ellens et al., 1974), is located at the right molecular end of the viral DNA. Yet, after short pulses of [3H]thymidine the relative yields of tritium in fragments D, E, and C were always low. Moreover, the relative yields of these three fragments of comparable size always constituted a very steep gradient in the order D > E > C, as it would be expected if the origin, or one of the origins, of replication was located at the left of D. Therefore, we feel our data clearly exclude displacement synthesis from a unique origin located at the right molecular end of the Ad. 2 genome. It would appear that our data also provide rather compelling evidence of a central location of the origin of replication in Ad. 2 DNA, a possibility that has already been envisaged by Winnacker (1974). After short pulse-labeling periods, the central fragments are the least labeled in mature molecules, while being the most labeled in pieces of all sizes derived from replicating molecules (Fig. 5). In replicating molecules, they are also the most frequently represented of all fragments. Such a central location for the origin of replication, however, cannot be reconciled easily with the electron microscopic observations made in several laboratories, including ours, on replicating adenovirus DNA. Specifically, it is difficult to see how a centrally initiated replication process would generate branched intermediates which all appear Y-shaped in the electron microscope (Sussenbach et al., 1972; Ellens et al., 1974; Bourgaux-Ramoisy et al., 1974). Yet, failure to observe the characteristic “eye-shaped” structures in preparations of replicating adenovirus DNA can hardly be regarded as strong evidence against the possibility of replication being actually in-
ADENOVIRUS
DNA
97
itiated from the center of the molecule. In this respect, it should be recalled that the concept of adenovirus DNA replication proceeding by displacement synthesis from a unique end of the molecule was based largely on electron microscopic observations (Sussenbach et al., 1972; Ellens et al., 1974). This concept was recently challenged by several independent groups on the basis of data obtained by restriction enzyme analysis and/or molecular hybridization of pulse-labeled viral DNA (Horwitz, 1974; Winnacker, 1974, Schilling et al., 1975; Tolun and Pettersson, 1975; Lavelle et al., 1975; this work). In the face of direct disagreement between electron microscopic observations and biochemical data, such as those presented herein suggesting a centrally-initiated process, we would therefore tend to favor the latter. At present, we are carrying out experiments with the aim of distinguishing between two alternate modes of replication, that directly supported by the data reported here, and another one, also implying termination at both molecular ends, namely initiation at both ends (Horwitz, 1974; Schilling et al., 1975). ACKNOWLEDGMENTS We thank Dr. E. Bradley for critical reading of the manuscript, and Miss Diane Bourbeau for technical assistance. This work was supported by grants from the Medical Research Council of Canada and from the National Cancer Institute of Canada. REFERENCES BELLETT, A. J. D., and YOUNGHU~BAND, H. B. (1972). Replication of the DNA of chick embryo lethal orphan virus. J. Mol. Biol. 72, 691-709. BOURGAUX, P., and BOURGAIJX-RAMOISY, D. (1971). A symmetrical model for polyoma virus DNA replication. J. Mol. Bid. 62, 513-524. BOURGAUX, P., and BOURGAUX-RAMOIEY, D. (1972). Unwinding of replicating polyoma virus DNA. J. Mol. Biol. 70, 399-413. BOURGAUX, P., BOURGAUX-RAMOISY, D., and SEILER, P. (1971). The replication of the ringshaped DNA of polyoma virus. II. Identification of molecules at various stages of replication. J. Mol. Biol. 59, 195-206. BOURGAUX-RAMOISY, D., ROBIN, J., and BOURGAUX, P. (1974). Replicating DNA of adenovirus type 2. Canad. J. Biochen. 52, 181-189. CRAWFORD, L. V., ROBBINS, A. K., NICXLIN, P. M., and OSBORN, K. (1974). Polyoma DNA replication:
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Location of the origin in different virus strains. Cold Spring Harbor Symp. Quant. Biol. 39, 219225. DANNA, K., and NATHANS, D. (1972). Bidirectional replication of simian virus 40 DNA. Proc. Nat. Acad. Sci. USA 69, 3097-3100. ELLENB, D. J., SUSSENBACH, J. S., and JANSZ, H. S. (19741 Studies on the mechanism of replication of adenovirus DNA. II. Electron microscopy of replicating DNA. Virology 61, 427-442. FAREED, G. C., GAEON, C. F., and SALZMAN, N. P. (19721. Origin and direction of simian virus 40 deoxyribonucleic acid replication. J. Virol. 10, 484-491. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cultures. J. Mol. Biol. 26, 365-369. HORWITZ, M. S. (1974). Location of the origin of DNA replication in adenovirus type 2. J. Virol. 13, 1046-1054. LAVELLE, G., PATCH, C., KHOURY, G., and ROSE, J. (1975). Isolation and partial characterization of single-stranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA. J. Viral. 16, 775-782. MULDER, C., ARRAND, J., DELIUS, H., KELLER, W., PETTER~SON, U., ROBERTS, R. J., and SHAIW, P. (1974). Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases EcoRI and Hpa I. Cold Spring Harbor Symp. Quunt. Biol. 39, 397-400. NATHANS, D., and DANNA, K. J. (1972). Specific origin in SV40 DNA replication. Nature New Biol.
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236, 200-202. PJYrTEzaSON, U. (1973). Some unusual properties of replicating adenovirus type 2 DNA. J. Mol. Biol. 81, 521-527. PE~TERSBON, U., MULDER, C., DELIUS, H., and SHARP, P. A. (1973). Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R.R1. PFOC.Nat. Acad. Sci. USA 70, 200-204. ROBIN, J., BOURGAUX-RAMOISY, D., and BOURGAUX, P. (1973). Single-stranded regions in replicating DNA of adenovirus type 2. J. Gen. Virol. 20, 233237. SCHILLING, R., WEING~RTNER, B., WINNACKER, E.L. (1975). Adenovirus type 2 DNA replication. II. Termini of DNA replication. J. Virol. 16,767-774. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373-390. SU~ENBACH, J. S., VAN DER VLIET, P. C., ELLENS, D. J., and JANSZ, H. S. (1972). Linear intermediates in the replication of adenovirus DNA. Nature New Biol. 239, 47-49. TOLUN, A., and PETTERBBON,U. (1975). Termination sites for adenovirus type 2 DNA replication. J. Viral. 16, 759-766. VAN DER EB, A. J. (1973). Intermediates in type 5 adenovirus DNA replication. Virology 51, 11-23. VINOG~AD, J., MORRIS, J., DAVIDSON, N., and DOVE, W. F., JR. (1963). The buoyant behavior of viral and bacterial DNA in alkaline CsCl. Proc. Nat. Acad. Sci. USA 49, 12-17. WINNACHER, E. L. (1974). Origins and termini of adenovirus type 2 DNA replication. Cold Spring Harbor Symp. Quant. Biol. 39, 547-550.