JouRNAL oF VntoLoGY, July, 1975, p. 17-26 Copyright 0 1975 American Society for Microbiology
Vol. 16, No. 1 Printed in U.S.A.
Intermediate in Adenovirus Type 2 Replication GEORGE D. PEARSON Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331 Received for publication 27 September 1974
Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with Si endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 106 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.
Adenoviruses replicate in the nuclei of infected cells. Viral DNA most likely exists in the nucleus as a nucleoprotein complex. Pearson and Hanawalt (13) separated nascent viral DNA, as a replication complex, from finished molecules. The kinetics of labeling indicated that the adenovirus complex was an intermediate in replication. Studies with three complementation groups of type 31 adenovirus temperature-sensitive (ts) mutants, all defective in the initiation of viral DNA synthesis, showed that the replication complex does not form at nonpermissive temperatures (19, 27). Yamashita and Green (34) recently isolated from adenovirus-infected cells a nuclear membrane complex which contains two virus-specified DNA binding proteins (30, 31), as well as endonuclease and DNA polymerase activities (T. Yamashita, M. Arens, and M. Green, personal communication). Nevertheless, the intranuclear site of viral replication has not yet been identified. Electron microscope autoradiography indicates that viral DNA synthesis occurs in the nucleoplasm, not in association with the nuclear envelope (20, 21). There is as yet little information about the composition of the adenovirus complex. I show in this paper that the complex contains replicating adenovirus chromosomes which differ in physical properties from mature molecules. These results confirm and extend earlier studies on adenovirus replication. In addition, an anal-
ysis of nascent chains in replicating molecules reveals size classes that are integral multiples of a unit 1,750 nucleotides long ('ho of an adenovirus strand). A possible relationship between replication units and the nucleoprotein organization of the adenovirus chromosome is discussed (J. Corden, H. M. Engelking, and G. Pearson, manuscript in preparation). Moreover, evidence for a maturation step in adenovirus replication is provided. (A preliminary report of this work was presented at the Fourth Tumor Virus Meeting at Cold Spring Harbor, N.Y., in August 1972.) MATERIALS AND METHODS Coil culture and synchronization. HeLa S, cells were grown in suspension culture using medium F-13 (Grand Island Biological Co.) supplemented with 7% fetal calf serum. Cells were synchronized with respect to DNA synthesis by two exposures for 20 h to 2 mM thymidine separated by a period of 12 h (13). Often cellular DNA was uniformly labeled with ["4C]thymidine (3 pM, 15 sCi/4smol) during this period. Adenovirus infection. Inocula for all experiments consisted of type 2 adenovirus purified in CsCl density gradients according to Doerfler (5). Cells were infected with 104 particles/cell as previously described
(13).
Isolation of the replication complex. The isolation of the adenovirus replication complex has been described in detail (13). In brief, nuclei from infected cells were digested with Pronase and sodium dodecyl sulfate. After shearing, the lysate was layered on a 17
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sucrose shelf gradient at 2 C. The detergent crystallizes in the cold. The gradient was centrifuged at 25,000 rpm for 135 min at 0 C in the SW27 rotor. The replication complex collects as a turbid detergent band on the 60% sucrose shelf. DNA extraction. Cells (107/ml) were digested with 1 mg of Pronase/ml (previously incubated for 1 h at 37 C at 5 mg/ml), 0.5% sodium dodecyl sulfate, and 0.01 M EDTA for 1 h at 37 C. DNA was extracted with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol) by blending in a Vortex mixer for 1 min at room temperature. The phases were separated by centrifugation at 12,000 x g for 10 min, and the. aqueous phase was retained. CsCl density gradient centrifugation. Gradients with an initial density of 1.710 were constructed by dissolving 7.90 g of CsCl in 6.5 g of liquid. To minimize the loss of DNA by adsorption to walls, gradients contained 0.1% sarkosyl and were centrifuged in polyallomer tubes. Centrifugation was for 36 h at 37,000 rpm at 20 C in the 5OTi angle rotor. Fractions (0.3 ml) were collected volumetrically from a pin hole in the bottom of the tube by pumping mineral oil in at the top. CsCl gradients containing 300 ug of ethidium bromide per ml were constructed by dissolving 6.70 g of CsCl in 7.25 g of liquid to give an initial density of 1.550. Sucrose gradient centrifugation. Sucrose gradients (5 to 20%, wt/vol; 30 ml) were formed in SW27 tubes. Gradients were centrifuged at 25,000 rpm and 20 C. The duration of the runs and the compositions of the individual gradients are detailed in the appropriate figure legends. Fractions (1 ml) were collected volumetrically as described above. Radioactivity determinations. 8H and "4C were analyzed as described previously (13). Isotope overlap calculations were computed on a Hewlett-Packard 9821A calculator. Materials. Pronase (free of nucleases), ethidium bromide, and Aquacide II were purchased from Calbiochem; pancreatic RNase (five times crystallized) and thymidine came from Schwarz/Mann; and [methyl- 14C Ithymidine was from New England Nuclear. The single-strand-specific S1 endonuclease from Aspergillus oryzae was purified from Enzopharm powder (Enzyme Development Corp.) and assayed as described by Sutton (28).
RESULTS Specific labeling of adenovirus DNA. HeLa cells were synchronized by two successive exposures to 2 mM thymidine and infected with type 2 adenovirus at the beginning of S phase. Infected cells proceeded normally through S phase, but did not enter mitosis nor initiate a subsequent round of cellular DNA synthesis (13, 21). Since cellular replication had ceased by 10 h after infection, adenovirus molecules could be labeled exclusively between 13 and 16 h after infection, the period of maximal viral DNA replication. All experiments reported here were started 13 h after infection.
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Demonstration of intermediate DNA in the replication complex. Figure 1A shows the distribution of a 6-min pulse of [3H ]thymidine in a sucrose shelf gradient. About 60 to 70% of the pulse label (newly replicated DNA) collected on the shelf as a detergent band (13). Less than 5% of the [4C ]cellular DNA sedimented onto the shelf; the bulk remained at the top of the gradient. The detergent band (labeled b) and the combined top fractions (marked c) were centrifuged separately to equilibrium in CsCl density gradients. Nascent viral DNA isolated from the detergent band had a buoyant density of 1.725 (Fig. 1B), greater than the density of marker adenovirus DNA (1.715; Fig. 1E). [14C]HeLa DNA banded at 1.700. Denatured adenovirus DNA has a density of 1.730 (25). Ten percent of the 3H label appeared as a shoulder tailing through the position of marker viral DNA. On the other hand, Fig. 1C shows that [3H ]DNA from the top fractions banded at the expected density of 1.715. No pulse label was found in the region of cellular DNA in either gradient. Newly replicated molecules exhibiting increased buoyant density can be extracted directly from infected cells after digestion with Pronase and detergent (Fig. 1D). DNA labeled for 6 min with [3H ]thymidine was broadly distributed with a peak at 1.725 and a prominent shoulder at 1.715. Viral DNA labeled for 1 h banded at the position of marker DNA with a shoulder at 1.725 (not shown). No radioactivity was detected at 1.725 when a 6-min pulse was chased for 90 min with 10-4 M unlabeled thymidine (not shown). Isolation of intermediate DNA in cesium chloride-ethidium bromide density gradients. Infected cells were labeled with [3H ]thymidine for 6 min. After extraction, the DNA was banded in a CsCl density gradient containing ethidium bromide (Fig. 2A). A major peak was observed at a density of 1.605. A minor peak at 1.585 coincided with the band of [14C ]HeLa DNA. Doerfler et al. (6, 7) have also shown that during infection some of the intracellular adenovirus DNA can be isolated as a dense band in propidium diiodide-cesium chloride gradients. The dense band (indicated by the bar in Fig. 2A) was further analyzed by velocity sedimentation in a neutral sucrose gradient (Fig. 2B). Over 85% of the radioactivity sedimented faster than 31S, the rate for marker adenovirus DNA. Some of the pulse sedimented faster than 60S (fractions marked as a). The rest of the pulse sedimented from 31 to 50S (labeled b). For comparison, the calculated
REPLICATING ADENOVIRUS DNA
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FIG. 1. Identification of intermediate DNA in the adenovirus replication complex. Synchronized, infected cells were concentrated to 1.6 x 101 cells/ml and labeled for 6 min with [3H]thymidine (10 iCi/ml at 20 Ci/mmol). Symbols: 0, [14CJHeLa DNA; 0, [sHjadenovirus DNA. (A) Cells (8 x 106) were converted to a nuclear lysate and centrifuged on a sucrose shelf gradient as previously described (13). Sedimentation is from right to left in all figures. The shelf fraction (b) and the fractions at the top of the gradient (c) were extracted with chloroform-isoamyl alcohol and concentrated by dialysis against solid Aquacide II. (B) Centrifugation of the shelf fraction (b) in a CsCI density gradient with an initial p = 1.710. Density increases from right to left in all figures. (C) CsCI density gradient analysis offractions at the top of the sucrose shelf gradient (c). (D) Cells (4 x 10') described above were extracted with chloroform-isoamyl alcohol after digestion with Pronase and sodium dodecyl sulfate. The whole-cell extract was centrifuged to equilibrium in a CsCI gradient. (E) ["1C]HeLa DNA from uninfected cells was mixed with [3HJadenovirus DNA extracted from purified viral particles and centrifuged to equilibrium in a CsCI gradient.
sedimentation coefficients for linear dimer and trimer molecules are 40 and 45S, respectively (22). Fractions a and b both had a buoyant density of 1.725 in CsCl (not shown). Figure 3A shows that after labeling intracellular adenovirus DNA for 15 min two prominent peaks appeared at densities of 1.605 and 1.585. [14C ]HeLa DNA again coincided with the lighter band (not shown). When centrifuged in CsCl gradients after removing the ethidium bromide, the dense peak (designated b) banded at a density of 1.725 (Fig. 3B) and the light peak (marked c) banded at 1.715 (Fig. 3C). Figure 3D shows that two peaks with the expected densities appeared when b and c were mixed. The dense band marked e, intermediate DNA, was broadly distributed in a neutral sucrose gradient (Fig. 3E). Only about one-half of the DNA sedimented faster than 31S, although some of the molecules still sedimented as fast as 60S.
The difference between gradient profiles in Fig. 2B and 3E most likely can be attributed to shearing due to the extra purification step. The small shoulder at 26S corresponds to duplex molecules one-half the molecular weight of adenovirus DNA. The light CsCl band, pooled as f, sedimented at 31S with a slight shoulder at 26S (Fig. 3F). Figure 3G demonstrates that a fast sedimenting peak at 42S appeared when a mixture of e and f were centrifuged together. Intermediate DNA sedimenting between 35 to 40S (fractions marked h in Fig. 3E) banded at a density of 1.725 (Fig. 3H). Intermediated DNA sedimenting near 31S (fractions labeled i in Fig. 3E) banded at a slightly lower density, spanning the region between 1.715 and 1.725 (Fig. 3I). Mature 31S DNA (peak j in Fig. 3F), previously isolated at a density of 1.715, remained at the same density (Fig. 3J). Kinetics of labeling intermediate DNA.
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FIG. 2. Isolation of intermediate DNA by equilibrium centrifugation in cesium chloride-ethidium bromide density gradients. Synchronized, infected cells were concentrated to 1.7 x 10" cells/ml and labeled for 6 min with [3H]thymidine (10 MCi/ml at 6.7 Ci/mmol). (A) Whole cells were extracted as described in Fig. 1 and centrifuged to equilibrium in a CsCl density gradient (initial p 1.550) containing 300 isg of ethidium bromide/ml. Symbols: 0, [14C]HeLa DNA; 0, ['H]adenovirus DNA. The fractions indicated by the bar were pooled, extracted with isopropanol to remove the ethidium bromide, and dialyzed to remove the isopropanol and CsCl. (B) Velocity sedimentation of intermediate DNA in a neutral sucrose gradient. The gradient contained 1.0 M NaCl, 0.005 M EDTA, and 0.01 M Tris, pH 8. Centrifugation was for 7 h at 25,000 rpm. The arrow indicates the position of marker viral DNA in a separate gradient (not shown). Fractions labeled a and b were pooled separately and further analyzed on CsCI density gradients (not shown). =
Replicating molecules should be labeled preferentially during intervals shorter than the time to synthesize completed daughter molecules. Figure 4A displays the distribution in cesium chloride-ethidium bromide gradients of DNA labeled with [3H ]thymidine for various periods of time. During the first minute, label appeared exclusively in intermediate DNA with a density of 1.605. The shortest pulse was 20 s (not shown). Incorporation into DNA banding at 1.585 required 3 to 5 min. The total incorporation in both peaks, normalized for the recovery of [14C ]HeLa DNA in each gradient, is presented in Fig. 4B as a function of the labeling time. Radioactivity was located predominantly in intermediate DNA during the first 10 min. After a short delay (extrapolated back to 3 min), label accumulated linearly in finished molecules. Figure 4B also shows that about 50% of the pulse label could be chased from intermediate DNA into completed viral molecules after adding a 1,000-fold excess of unlabeled thymidine at 10 min. The chase was effective within 10 min (Fig. 4B, inset). The kinetics of labeling
and the chase confirm a precursor-product relationship. Adenovirus DNA is replicated in 17 min, the time required to label intermediate DNA and completed molecules equally (i.e., the intersection in Fig. 4B). Intermediate DNA and completed viral molecules from each time point were also analyzed by velocity sedimentation in alkaline sucrose gradients (Fig. 5A through I). In agreement with Horwitz (9), no single strands longer than unit length (34S) were ever detected. After 1 min of labeling, strands of intermediate DNA (Fig. 5A) sedimented in the region of lOS (1,750 nucleotides) to 14S (3,500 nucleotides) with faster sedimenting shoulders at 19S (9,000 nucleotides) and 26S (17,500 nucleotides). Chains as short as 400 nucleotides could also be detected. Vlak et al. (33) and E. Winnacker (personal communication) have also found 9 to 11S strands as intermediates in adenovirus replication. Since the lOS chain corresponds to 'ho of an adenovirus strand, I have defined this unit as a "faceful" (i.e., equivalent to one of the 20 faces of the icosahedral virus particle). The signifi-
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cance of facefuls will be discussed below. After 2 (Fig. 5B) or 3 min (Fig. 5C) of labeling, strands of intermediate DNA sedimented at 14, 18, 23, and 26S, which correspond to 2, 4, 8, and 10 facefuls. Longer strands of 30 to 32S (16 to 18 facefuls) appear by 4 (Fig. 5D) and 5 min (Fig. 5E). The size of nascent chains reached an equilibrium distribution of 26S within 10 min (Fig. 5F). Unit-length single strands were also present at this time. Further experiments to size nascent strands by agarose gel electrophoresis are in progress. At all times, completed molecules sedimented primarily as unit-length single strands. Some newly finished molecules evidently have transient single-strand interruptions. Finished molecules isolated after 10 (Fig. 5G) or 15 min (Fig. 5H) of labeling contain shorter fragments sedimenting at 18, 23, 26, and 31S (i.e., 1/4, 1/3, 1/2, and 3/4 fractional lengths). After a chase with unlabeled thymidine for 80 min (Fig. 5I), all strands were unit length. This also excludes
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the possiblity that ethidium bromide introduced strand breaks during the separation of intermediate DNA from finished molecules. The center of mass of each sucrose gradient profile of intermediate DNA was plotted in Fig. 5J as a function of labeling time. Interestingly, the graph extrapolated to a molecular weight of 5.8 x 10' (or 1,750 nucleotides) for an infinitely short pulse. The initial rate of chain elongation was 0.7 x 106 daltons/min. The time to complete unit-length single strands was calculated to be 16 min, in close agreement with the estimate in Fig. 4B. Properties of intermediate DNA. Either single-stranded DNA or RNA could increase the buoyant density of intermediate DNA. Pettersson (14) showed that the density difference between replicating and mature adenovirus DNA was eliminated after digestion with the single-strand-specific nuclease from Neurospora crassa, but not after digestion with RNase. The following experiments confirm
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0 10 l0 20 30 20 30 l0 20 30 FRACTIONS FRACTIONS FRACTIONS FiG. 3. Characterization of intermediate DNA and completed viral DNA. A sample from the culture described in Fig. 2 was removed after labeling for 15 min. (A) Equilibrium centrifugation of extracted DNA in an ethidium bromide-cesium chloride density gradient. Fractions labeled b and c were pooled separately, extracted with isopropanol, and dialyzed. ["4C]HeLa DNA (not shown) co-banded with the light band (c). (B) Rebanding of fraction b and ["4CJHeLa DNA (not shown) in a neutral CsCI density gradient (initial p 1.710). Fractions labeled e were pooled and dialyzed. The vertical dashed lines represent, from left to right: intermediate DNA, adenovirus DNA, and HeLa DNA. (C) Rebanding of fraction c and ["4ClHeLa (not shown) in a neutral CsCI density gradient. Fractions labeled f were pooled and dialyzed. (D) Rebanding of a mixture of b and c in a neutral CsCI density gradient. Closed circles represent marker ["4C]HeLa DNA. (E) Neutral sucrose gradient velocity sedimentation of fraction e. Sucrose gradients (containing 1.0 M NaCI, 0.005 M EDTA, and 0.01 M Tris, pH 8) were centrifuged for 7 h at 25,000 rpm. Fractions labeled h and i were pooled separately and dialyzed. (F) Neutral sucrose gradient velocity sedimentation offraction f. The fraction labeled j was retained and dialyzed. (G) Neutral sucrose gradient velocity sedimentation of a mixture of e and f. The arrow marks the position of 42S. (H) Rebanding of fraction h in a neutral CsCI density gradient. Symbols: *, [CJC]HeLa DNA. The vertical dashed lines represent, from left to right: intermediate DNA, adenovirus DNA, and HeLa DNA. (1) Rebanding offraction i and ["4C JHeLa DNA (not shown) in a neutral CsCI density gradient. (J) Rebanding of fraction j and ["C JHeLa DNA (not shown) in a neutral CsCI density gradient. 0
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FIG. 4. Kinetics of labeling intermediate DNA and finished adenovirus DNA. Infected cells were concentrated to 4 x 106 cells/ml and labeled with [9H]thymidine (10 tiCi/ml at 10 Ci/mmol). At 10 min the incorporation of [3H]thymidine was quenched by adding 10-4 M unlabeled thymidine. Samples of 4 x 106 cells were removed at the indicated times, extracted, and centrifuged to equilibrium in cesium chloride-ethidium bromide gradients. (A) Profiles of cesium chloride-ethidium bromide density gradients. The peak of [14C]HeLa DNA (not shown) is indicated by the arrow. The [9H]thymidine incorporated into adenovirus DNA was normalized to the recovery of HeLa DNA in each gradient (45,000 counts/min of 14C). Symbols: 0, 1 min; 0, 3 min; A, 5 min; 0, 10 min. (B) Radioactivity in the peak fractions of the cesium chloride-ethidium bromide gradients was normalized for recovery of HeLa DNA in each gradient and plotted as a function of the time of labeling. The chase with 10-4 M thymidine was started 10 min after adding the [3H]thymidine. Symbols: 0, 3H in intermediate DNA; 0, 3H in completed adenovirus DNA. (Insert) Total incorporation of. ['H]thymidine in each gradient as a function of time. The dashed line indicates the initial rate of incorporation. The arrow indicates the beginning of the chase.
those observations. Purified intermediate DNA, labeled for 5 min with ['H ]thymidine, was mixed with [14C ]adenovirus DNA and digested with pancreatic RNase or the single-strandspecific Sl endonuclease from A. oryzae (1, 28). Intermediate DNA had a buoyant density of 1.725 (Fig. 6A). After treatment with Si endonuclease (Fig. 6B), intermediate DNA banded at the density of marker DNA, although less than 1% of 3H and '4C labels were solubilized. Under the same conditions, more than 95% of heat-denatured viral DNA could be digested. Figure 6C shows that RNase digestion in buffer containing 0.2 M NaCl had no effect on the density of intermediate DNA. RNase hydrolyzed better than 98% of ['H ]HeLa rRNA under the same conditions. These experiments strongly suggest that the increased buoyant density of intermediate DNA is due to substan-
tial single strands of parental DNA. In this regard, replicating adenovirus chromosomes appear in electron micrographs as branched and unbranched linear molecules with extensive single-stranded regions (8, 23, 26, 29). Thus, adenovirus might replicate by displacement synthesis, a mechanism proposed for mitochondrial DNA replication (3, 16, 17). In spite of the above conclusions, hydrogenbonded RNA may play a role in maintaining the structure of intermediate DNA. Figure 6D demonstrates that RNase disrupted intermediate DNA in 0.02 M NaCl, conditions where RNase attacks RNA in RNA-DNA hybrids. In three experiments an average of 9% (range, 6 to 12%) of the 3H label banded at the position of marker DNA. The rest of the 3H activity floated at the meniscus of the CsCl gradient (the total recovery of 3H was better than 70%). An interpreta-
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REPLICATING ADENOVIRUS DNA
tion is that nascent strands in intermediate DNA are released as a low density complex. The complex must be insensitive to detergents, Pronase, and concentrated salt solutions. Pettersson (14) also reported that RNase digestion in buffer lacking salt reduces the buoyant density of replicating adenovirus DNA, but the magnitude of the change was less than reported here. Furthermore, intermediate DNA can be labeled with radioactive uridine (7; G. D. Pearson, unpublished data). The sedimentation rate of intermediate DNA is markedly altered by changes in ionic strength. Figure 7A demonstrates that intermediate DNA formed a fast sedimenting aggregate (greater than 100S) when the ionic strength was lowered to 0.01 M NaCl. At salt concentrations greater than 0.1 M NaCl, intermediate DNA sedimented as a broad zone with peaks at 47 and 31S (Fig. 7B; compare with Fig. 2B and
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3E). The sedimentation rate of marker adenovirus DNA was not affected within the range of ionic strengths used in these experiments. It is difficult to attribute this behavior to singlestranded regions in intermediate DNA. Single DNA chains assume an extended conformation in low salt due to electrostatic repulsion by negatively charged phosphates. The sedimentation rate decreases as a consequence (22). Although the basis for the aggregation is not known, it has been used to separate finished molecules from replicating molecules to construct a replication map of adenovirus (G. D. Pearson, manuscript in preparation) by the method of Danna and Nathans (4). DISCUSSION Nascent DNA contained in the adenovirus replication complex, called intermediate DNA, was shown to differ physically from mature viral
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10 l0 20 30 l0 20 30 FRACTIONS FRACTIONS FRACTIONS FIG. 5. Alkaline sucrose gradient velocity sedimentation of intermediate DNA and completed adenovirus DNA isolated from the cesium chloride-ethidium bromide gradients described in Fig. 4. Peak fractions were pooled separately, extracted with isopropanol, and dialyzed. The fractions were then denatured by adjusting to 0.2 M NaOH and centrifuged for 7 h at 25,000 rpm in alkaline sucrose gradients (containing 1.0 M Na+). At least 2,000 counts/min of 3H were in each gradient. (A) Intermediate DNA, 1-min pulse; (B) intermediate DNA, 2-min pulse; (C) intermediate DNA, 3-min pulse; (D) intermediate DNA, 4-min pulse; (E) intermediate DNA, 5-min pulse; (F) intermediate DNA, 10-min pulse; (G) completed viral DNA, 10-min pulse; (H) completed viral DNA, 10-min pulse plus 5-min chase; (I) completed viral DNA, 10-min pulse plus 80-min chase; (J) plot of the center of mass of intermediate DNA in each gradient as a function of the labeling time. The center of mass was calculated from the relationship . ,f1CIC,, where f, fraction number and Cl counts per minute in that fraction. This was converted to single-stranded molecular weight (22). 0
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FRACTIONS F I G. 6. Digestion of intermediate DNA with Sl endonuclease or ribonuclease. ['H]intermediate DNA was purified as described in Fig. 2, For Sl endonuclease, the reaction contained per 0.3 ml: 25 Ml of ['HJintermediate DNA; 10 MI of ['4C]adenovirus DNA; 7.5 Mg of heat-denatured calf thymus DNA; 25 MI of enzyme in 0.07 M NaCl, 0.03 M sodium citrate, 0.001 M ZnCI2, pH 4.5. The mixture was incubated for 1 h at 50 C, stopped by adding 0.5% sarkosyl, and centrifuged in CsCI gradients containing 0.5% sarkosyl. RNase was first heated to 80 C for 10 min. Reactions in high salt contained per 1.0 ml: 25 Al of [(H]intermediate DNA, 10 Al of ['4C]adenovirus DNA, and 100 Mg ofpancreatic RNase in 0.2 MNaCI, 0.01 M EDTA, 0.01 M Tris, pH8. After I h at 37 C, the reactions were stopped by adding 0.5% sarkosyl and centrifuged as above. Reactions in low salt were identical except that the buffer contained: 0.02 M NaCI, 0.01 M EDTA, 0.01 M Tris, pH 8. Symbols: 0, [3H]intermediate DNA; 0, ['4C]adenovirus DNA. (A) No Sl endonuclease (control). The controls in high and low salt without RNase gave similar profiles (not shown); (B) with Sl endonuclease; (C) with pancreatic RNase in 0.2 M NaCI; (D) with pancreatic RNase in 0.02 M NaCI.
molecules. For example, intermediate DNA had an increased buoyant density (1.725 compared to 1.715) and a greater sedimentation coefficient (up to 100S compared to 31S). Since treatment with the single-strand-specific Si endonuclease, but not with RNase, lowered the buoyant density of intermediate DNA without loss of the pulse label, a substantial fraction of the parental strands must be single stranded (also see 14). Branched and unbranched linear molecules containing extensive single-stranded regions have been visualized in the electron microscope (8, 23, 26, 29). These properties agree with the properties of replicating adenovirus DNA reported by other laboratories (2, 8, 23-26, 29, 32). An improved method for isolating intermediate DNA has been developed. Ethidium bromidecesium chloride density gradients provide increased separation between intermediate DNA (1.605) and mature viral DNA (1.585). Experiments reported here carefully document the identities of these bands. When labeled for 1 min or less, nascent strands from intermediate DNA sedimented
primarily at lOS, corresponding to a chain 1,750 nucleotides long. No strands longer than an intact viral strand were ever detected. Vlak et al. (33) and E. Winnacker (personal communication) have shown that 9 to 11S nascent chains are complementary to both viral strands throughout the entire genome. Remarkably, other nascent strands at this and longer times of labeling were integral multiples of 1,750 nucleotides. This replication unit has been defined as a faceful, since it is exactly '/2 of a finished adenovirus strand. Recent experiments suggest a relationship between replication units and the nucleoprotein structure of the virus chromosome. Double-stranded equivalents of the faceful (i.e., 1,750 base pairs) appear as the initial product when disrupted adenovirus particles are digested with staphylococcal nuclease. Longer digestion times yield homogeneous fragments of about 150 base pairs. We postulate that a regular arrangement of core proteins protect discrete regions of viral DNA from nuclease attack. A complete model with supporting evidence will be published elsewhere (J.
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FRACTIONS FIG. 7. Effect of ionic strength on the sedimentation rate of intermediate DNA. ['H]intermediate DNA was mixed with ["ICfadenovirus DNA and sedimented for 5 h at 25,000 rpm in neutral sucrose gradients containing 0.01 M NaCI, 0.Ou M EDTA, and 0.01 M Tris, pH8 (A), or U.1 M NaCI, 0.001 M EDTA, and 0.01 M Tris, pH 8 (B). The prorfue o0 intermediate DNA in gradients containing 1.0 M NaCI, 0.001 M EDTA, and 0.01 M Tris, pH 8, resembled the profile in (B). Symbols: 0, ['H]intermediate DNA; *, [(1C adenovirus DNA.
Corden, H. M. Engelking, and G. Pearson, manuscript in preparation). The average time required for adenovirus replication was calculated to be 16 to 17' min, corresponding to a rate of 0.7 x 106 daltons/min for chain elongation. This rate compares favorably with 0.5 x 106 daltons/min for chicken embryo lethal orphan virus, an avian adenovirus (2), 0.6 x 106 daltons/min for simian virus 40 (12), and 0.8 x 106 daltons/min for the L strand of mitochondrial DNA (3). Pearson and Hanawalt (13) previously overestimated the replication rate because excessive shear was used to isolate the replication complex. Although newly finished molecules sedimented at 31S and had a density of 1.715, they still contained single-strand interruptions. Mature chromosomes do not. Complete joining of daughter strands required at least an additional 15 to 20 min (unpublished observations). Unjoined strands were approximately 0.25, 0.5, and 0.75 fractional lengths. Interestingly, an adenovirus replication map (Pearson, manuscript in preparation), constructed by the method of Danna and Nathans (4), locates an origin in Eco RI fragment F or in Eco RI fragment B very near to fragment F and at least
one other origin within the Eco RI fragment A in a region defined by the Hpa I fragment F (E. Winnacker, personal communication). The sizes (15) and order (11) of the Eco RI endonuclease fragments of adenovirus DNA have been reported. Thus, two replicative origins are positioned about 25% from either end of the chromosome. Horwitz (10) also has demonstrated origins in both halves of the adenovirus genome using a similar approach. Unbranched molecules with many single-stranded gaps have been visualized by electron microscopy (8, 26, 29). It is tempting to speculate that unjoined strands are sealed at the junctions between origins and termini. ACKNOWLEDGMENTS This research was supported by grants (NP-67, NP-67A) from the American Cancer Society and a Biomedical Sciences Support Grant (5 S05 RR07079-06) administered through the Oregon State University Research Council. Part of this work was done as a Dernham Junior Fellow (J-126) of the California Division of the American Cancer Society. I thank Philip Hanawalt, John Kiger, Ernst Winnacker, David Dressler, and John Wolfson for many valuable suggestions and discussions. I also thank John Sussenbach for a copy of his paper prior to publication. Gail Foster provided devoted technical assistance.
26
PEARSON LITERATURE CITED
1. Ando, T. 1966. A nuclease specific for heat-denatured DNA isolated from a product of Aspergillus oryzae. Biochem. Biophys. Acta 114:158-168. 2. Bellett, 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. Berk, A. J., and D. A. Clayton. 1974. Mechanism of mitochondrial DNA replication in mouse L-cells; asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence. J. Mol. Biol. 86:801-824. 4. Danna, K. J., and D. Nathans. 1972. Bidirectional replication of simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 69:3097-3100. 5. Doerfler, W. 1969. Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology 38:587-606. 6. Doerfler, W., U. Lundholm, and M. Hirsch-Kauffmann. 1972. Intracellular forms of adenovirus deoxyribonucleic acid. I. Evidence for a deoxyribonucleic acid protein complex in baby hamster kidney cells infected with adenovirus type 12. J. Virol. 9:297-308. 7. Doerfler, W., U. Lundholm, U. Rensing, and L. Philipson. 1973. Intracellular forms of adenovirus DNA. II. Isolation in dye-buoyant density gradients of a DNA-RNA complex from KB cells infected with adenovirus type 2. J. Virol. 12:793-807. 8. Ellens, D. J., J. S. Sussenbach, and H. S. Jansz. 1974. Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA. Virology 61:427-442. 9. Horwitz, M. S. 1971. Intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid. J. Virol. 8:675-683. 10. Horwitz, M. S. 1974. Location of the origin of DNA replication in adenovirus type 2. J. Virol. 13:1046-1054. 11. Mulder, C., P. A. Sharp, H. Delius, and U. Pettersson. 1974. Specific fragmentation of DNA of adenovirus serotypes 3, 5, 7, and 12, and adeno-simian virus 40 hybrid virus Ad2+ND1 by restriction endonuclease R-EcoRI. J. Virol. 14:68-77. 12. Nathans, D., and K. J. Danna. 1972. Specific origin in SV40 DNA replication. Nature (London) New Biol. 236:200-202. 13. Pearson, G. D., and P. C. Hanawalt. 1971. Isolation of, DNA replication complexes from uninfected and adenovirus-infected HeLa cells. J. Mol. Biol. 62:65-80. 14. Pettersson, U. 1973. Some unusual properties of replicating adenovirus type 2 DNA. J. Mol. Biol. 81:521-527. 15. Pettersson, U., C. Mulder, H. Delius, and P. Sharp. 1973. Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R -RI. Proc. Natl. Acad. Sci. U.S.A. 70:200-204. 16. Robberson, D. L., and D. A. Clayton. 1972. Replication of mitochondrial DNA in mouse L cells and their thymidine kinase derivatives: displacement replication on a covalently-closed circular template. Proc. Natl. Acad.
J. VIROL. Sci. U.S.A. 69:3810-3814. 17. Robberson, D. L., and D. A. Clayton. 1973. Pulse-labeled components in the replication of mitochondrial deoxyribonucleic acid. J. Biol. Chem. 248:4512-4514. 18. Robinson, A. J., H. B. Younghusband, and A. J. D. Bellett. 1973. A circular DNA-protein complex from adenoviruses. Virology 56:54-69. 19. Shiroki, K., and H. Shimojo. 1974. Analysis of adenovirus 12 temperature-sensitive mutants defective in viral DNA replication. Virology 61:474-485. 20. Shiroki, K., H. Shimojo, and K. Yamaguchi. 1974. The viral DNA replication complex of adenovirus 12. Virology 60:192-199. 21. Simmons, T., P. Heywood, and L. D. Hodge. 1974. Intranuclear site of replication of adenovirus DNA. J. Mol. Biol. 89:423-433. 22. Studier, F. W. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390. 23. Sussenbach, J. S., D. J. Ellens, and H. S. Jansz. 1973. Studies on the mechanism of replication of adenovirus DNA. J. Virol. 12:1131-1138. 24. Sussenbach, J. S., and P. C. van der Vliet. 1972. Viral DNA synthesis in isolated nuclei from adenovirusinfected KB cells. FEBS Lett. 21:7-10. 25. Sussenbach, J. S., and P. C. van der Vliet. 1973. Studies on the mechanism of replication of adenovirus DNA. I. The effect of hydroxyurea. Virology 54:299-303. 26. Sussenbach, J. S., P. C. van der Vliet, D. J. Ellens, and H. S. Jansz. 1972. Linear intermediates in the replication of adenovirus DNA. Nature (London) New Biol. 239:47-49. 27. Suzuki, E., and H. Shimojo. 1974. Temperature-sensitive formation of the DNA replication complex in adenovirus 31-infected cells. J. Virol. 13:538-540. 28. Sutton, W. D. 1971. A crude nuclease preparation suitable for use in DNA reassociation experiments. Biochim. Biophys. Acta 240:522-531. 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 A. J. Levine. 1972. DNA-binding proteins specific for cells infected by adenovirus. Nature (London) New Biol. 246:170-174. 31. van der Vliet, P. C., A. J. Levine, M. J. Ensinger, and H. S. Ginsberg. 1975. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Virol. 15:348-354. 32. 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. 33. Vlak, J. M., T. H. Rozijn, and J. S. Sussenbach. 1975. Studies on the mechanism of replication of adenovirus DNA. IV. Discontinuous DNA chain propagation. Virology 63:168-175. 34. Yamashita, T., and M. Green. 1974. Adenovirus DNA replication. I. Requirement for protein synthesis and isolation of nuclear membrane fractions containing newly synthesized viral DNA and proteins. J. Virol. 14:412-420.
Vol. 16, No. 1 Printed in U.S.A.
Intermediate in Adenovirus Type 2 Replication GEORGE D. PEARSON Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331 Received for publication 27 September 1974
Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with Si endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 106 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.
Adenoviruses replicate in the nuclei of infected cells. Viral DNA most likely exists in the nucleus as a nucleoprotein complex. Pearson and Hanawalt (13) separated nascent viral DNA, as a replication complex, from finished molecules. The kinetics of labeling indicated that the adenovirus complex was an intermediate in replication. Studies with three complementation groups of type 31 adenovirus temperature-sensitive (ts) mutants, all defective in the initiation of viral DNA synthesis, showed that the replication complex does not form at nonpermissive temperatures (19, 27). Yamashita and Green (34) recently isolated from adenovirus-infected cells a nuclear membrane complex which contains two virus-specified DNA binding proteins (30, 31), as well as endonuclease and DNA polymerase activities (T. Yamashita, M. Arens, and M. Green, personal communication). Nevertheless, the intranuclear site of viral replication has not yet been identified. Electron microscope autoradiography indicates that viral DNA synthesis occurs in the nucleoplasm, not in association with the nuclear envelope (20, 21). There is as yet little information about the composition of the adenovirus complex. I show in this paper that the complex contains replicating adenovirus chromosomes which differ in physical properties from mature molecules. These results confirm and extend earlier studies on adenovirus replication. In addition, an anal-
ysis of nascent chains in replicating molecules reveals size classes that are integral multiples of a unit 1,750 nucleotides long ('ho of an adenovirus strand). A possible relationship between replication units and the nucleoprotein organization of the adenovirus chromosome is discussed (J. Corden, H. M. Engelking, and G. Pearson, manuscript in preparation). Moreover, evidence for a maturation step in adenovirus replication is provided. (A preliminary report of this work was presented at the Fourth Tumor Virus Meeting at Cold Spring Harbor, N.Y., in August 1972.) MATERIALS AND METHODS Coil culture and synchronization. HeLa S, cells were grown in suspension culture using medium F-13 (Grand Island Biological Co.) supplemented with 7% fetal calf serum. Cells were synchronized with respect to DNA synthesis by two exposures for 20 h to 2 mM thymidine separated by a period of 12 h (13). Often cellular DNA was uniformly labeled with ["4C]thymidine (3 pM, 15 sCi/4smol) during this period. Adenovirus infection. Inocula for all experiments consisted of type 2 adenovirus purified in CsCl density gradients according to Doerfler (5). Cells were infected with 104 particles/cell as previously described
(13).
Isolation of the replication complex. The isolation of the adenovirus replication complex has been described in detail (13). In brief, nuclei from infected cells were digested with Pronase and sodium dodecyl sulfate. After shearing, the lysate was layered on a 17
18
PEARSON
sucrose shelf gradient at 2 C. The detergent crystallizes in the cold. The gradient was centrifuged at 25,000 rpm for 135 min at 0 C in the SW27 rotor. The replication complex collects as a turbid detergent band on the 60% sucrose shelf. DNA extraction. Cells (107/ml) were digested with 1 mg of Pronase/ml (previously incubated for 1 h at 37 C at 5 mg/ml), 0.5% sodium dodecyl sulfate, and 0.01 M EDTA for 1 h at 37 C. DNA was extracted with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol) by blending in a Vortex mixer for 1 min at room temperature. The phases were separated by centrifugation at 12,000 x g for 10 min, and the. aqueous phase was retained. CsCl density gradient centrifugation. Gradients with an initial density of 1.710 were constructed by dissolving 7.90 g of CsCl in 6.5 g of liquid. To minimize the loss of DNA by adsorption to walls, gradients contained 0.1% sarkosyl and were centrifuged in polyallomer tubes. Centrifugation was for 36 h at 37,000 rpm at 20 C in the 5OTi angle rotor. Fractions (0.3 ml) were collected volumetrically from a pin hole in the bottom of the tube by pumping mineral oil in at the top. CsCl gradients containing 300 ug of ethidium bromide per ml were constructed by dissolving 6.70 g of CsCl in 7.25 g of liquid to give an initial density of 1.550. Sucrose gradient centrifugation. Sucrose gradients (5 to 20%, wt/vol; 30 ml) were formed in SW27 tubes. Gradients were centrifuged at 25,000 rpm and 20 C. The duration of the runs and the compositions of the individual gradients are detailed in the appropriate figure legends. Fractions (1 ml) were collected volumetrically as described above. Radioactivity determinations. 8H and "4C were analyzed as described previously (13). Isotope overlap calculations were computed on a Hewlett-Packard 9821A calculator. Materials. Pronase (free of nucleases), ethidium bromide, and Aquacide II were purchased from Calbiochem; pancreatic RNase (five times crystallized) and thymidine came from Schwarz/Mann; and [methyl- 14C Ithymidine was from New England Nuclear. The single-strand-specific S1 endonuclease from Aspergillus oryzae was purified from Enzopharm powder (Enzyme Development Corp.) and assayed as described by Sutton (28).
RESULTS Specific labeling of adenovirus DNA. HeLa cells were synchronized by two successive exposures to 2 mM thymidine and infected with type 2 adenovirus at the beginning of S phase. Infected cells proceeded normally through S phase, but did not enter mitosis nor initiate a subsequent round of cellular DNA synthesis (13, 21). Since cellular replication had ceased by 10 h after infection, adenovirus molecules could be labeled exclusively between 13 and 16 h after infection, the period of maximal viral DNA replication. All experiments reported here were started 13 h after infection.
J. VIROL.
Demonstration of intermediate DNA in the replication complex. Figure 1A shows the distribution of a 6-min pulse of [3H ]thymidine in a sucrose shelf gradient. About 60 to 70% of the pulse label (newly replicated DNA) collected on the shelf as a detergent band (13). Less than 5% of the [4C ]cellular DNA sedimented onto the shelf; the bulk remained at the top of the gradient. The detergent band (labeled b) and the combined top fractions (marked c) were centrifuged separately to equilibrium in CsCl density gradients. Nascent viral DNA isolated from the detergent band had a buoyant density of 1.725 (Fig. 1B), greater than the density of marker adenovirus DNA (1.715; Fig. 1E). [14C]HeLa DNA banded at 1.700. Denatured adenovirus DNA has a density of 1.730 (25). Ten percent of the 3H label appeared as a shoulder tailing through the position of marker viral DNA. On the other hand, Fig. 1C shows that [3H ]DNA from the top fractions banded at the expected density of 1.715. No pulse label was found in the region of cellular DNA in either gradient. Newly replicated molecules exhibiting increased buoyant density can be extracted directly from infected cells after digestion with Pronase and detergent (Fig. 1D). DNA labeled for 6 min with [3H ]thymidine was broadly distributed with a peak at 1.725 and a prominent shoulder at 1.715. Viral DNA labeled for 1 h banded at the position of marker DNA with a shoulder at 1.725 (not shown). No radioactivity was detected at 1.725 when a 6-min pulse was chased for 90 min with 10-4 M unlabeled thymidine (not shown). Isolation of intermediate DNA in cesium chloride-ethidium bromide density gradients. Infected cells were labeled with [3H ]thymidine for 6 min. After extraction, the DNA was banded in a CsCl density gradient containing ethidium bromide (Fig. 2A). A major peak was observed at a density of 1.605. A minor peak at 1.585 coincided with the band of [14C ]HeLa DNA. Doerfler et al. (6, 7) have also shown that during infection some of the intracellular adenovirus DNA can be isolated as a dense band in propidium diiodide-cesium chloride gradients. The dense band (indicated by the bar in Fig. 2A) was further analyzed by velocity sedimentation in a neutral sucrose gradient (Fig. 2B). Over 85% of the radioactivity sedimented faster than 31S, the rate for marker adenovirus DNA. Some of the pulse sedimented faster than 60S (fractions marked as a). The rest of the pulse sedimented from 31 to 50S (labeled b). For comparison, the calculated
REPLICATING ADENOVIRUS DNA
VOL. 16, 1975
19
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FIG. 1. Identification of intermediate DNA in the adenovirus replication complex. Synchronized, infected cells were concentrated to 1.6 x 101 cells/ml and labeled for 6 min with [3H]thymidine (10 iCi/ml at 20 Ci/mmol). Symbols: 0, [14CJHeLa DNA; 0, [sHjadenovirus DNA. (A) Cells (8 x 106) were converted to a nuclear lysate and centrifuged on a sucrose shelf gradient as previously described (13). Sedimentation is from right to left in all figures. The shelf fraction (b) and the fractions at the top of the gradient (c) were extracted with chloroform-isoamyl alcohol and concentrated by dialysis against solid Aquacide II. (B) Centrifugation of the shelf fraction (b) in a CsCI density gradient with an initial p = 1.710. Density increases from right to left in all figures. (C) CsCI density gradient analysis offractions at the top of the sucrose shelf gradient (c). (D) Cells (4 x 10') described above were extracted with chloroform-isoamyl alcohol after digestion with Pronase and sodium dodecyl sulfate. The whole-cell extract was centrifuged to equilibrium in a CsCI gradient. (E) ["1C]HeLa DNA from uninfected cells was mixed with [3HJadenovirus DNA extracted from purified viral particles and centrifuged to equilibrium in a CsCI gradient.
sedimentation coefficients for linear dimer and trimer molecules are 40 and 45S, respectively (22). Fractions a and b both had a buoyant density of 1.725 in CsCl (not shown). Figure 3A shows that after labeling intracellular adenovirus DNA for 15 min two prominent peaks appeared at densities of 1.605 and 1.585. [14C ]HeLa DNA again coincided with the lighter band (not shown). When centrifuged in CsCl gradients after removing the ethidium bromide, the dense peak (designated b) banded at a density of 1.725 (Fig. 3B) and the light peak (marked c) banded at 1.715 (Fig. 3C). Figure 3D shows that two peaks with the expected densities appeared when b and c were mixed. The dense band marked e, intermediate DNA, was broadly distributed in a neutral sucrose gradient (Fig. 3E). Only about one-half of the DNA sedimented faster than 31S, although some of the molecules still sedimented as fast as 60S.
The difference between gradient profiles in Fig. 2B and 3E most likely can be attributed to shearing due to the extra purification step. The small shoulder at 26S corresponds to duplex molecules one-half the molecular weight of adenovirus DNA. The light CsCl band, pooled as f, sedimented at 31S with a slight shoulder at 26S (Fig. 3F). Figure 3G demonstrates that a fast sedimenting peak at 42S appeared when a mixture of e and f were centrifuged together. Intermediate DNA sedimenting between 35 to 40S (fractions marked h in Fig. 3E) banded at a density of 1.725 (Fig. 3H). Intermediated DNA sedimenting near 31S (fractions labeled i in Fig. 3E) banded at a slightly lower density, spanning the region between 1.715 and 1.725 (Fig. 3I). Mature 31S DNA (peak j in Fig. 3F), previously isolated at a density of 1.715, remained at the same density (Fig. 3J). Kinetics of labeling intermediate DNA.
20
J. VIROL.
PEARSON
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FIG. 2. Isolation of intermediate DNA by equilibrium centrifugation in cesium chloride-ethidium bromide density gradients. Synchronized, infected cells were concentrated to 1.7 x 10" cells/ml and labeled for 6 min with [3H]thymidine (10 MCi/ml at 6.7 Ci/mmol). (A) Whole cells were extracted as described in Fig. 1 and centrifuged to equilibrium in a CsCl density gradient (initial p 1.550) containing 300 isg of ethidium bromide/ml. Symbols: 0, [14C]HeLa DNA; 0, ['H]adenovirus DNA. The fractions indicated by the bar were pooled, extracted with isopropanol to remove the ethidium bromide, and dialyzed to remove the isopropanol and CsCl. (B) Velocity sedimentation of intermediate DNA in a neutral sucrose gradient. The gradient contained 1.0 M NaCl, 0.005 M EDTA, and 0.01 M Tris, pH 8. Centrifugation was for 7 h at 25,000 rpm. The arrow indicates the position of marker viral DNA in a separate gradient (not shown). Fractions labeled a and b were pooled separately and further analyzed on CsCI density gradients (not shown). =
Replicating molecules should be labeled preferentially during intervals shorter than the time to synthesize completed daughter molecules. Figure 4A displays the distribution in cesium chloride-ethidium bromide gradients of DNA labeled with [3H ]thymidine for various periods of time. During the first minute, label appeared exclusively in intermediate DNA with a density of 1.605. The shortest pulse was 20 s (not shown). Incorporation into DNA banding at 1.585 required 3 to 5 min. The total incorporation in both peaks, normalized for the recovery of [14C ]HeLa DNA in each gradient, is presented in Fig. 4B as a function of the labeling time. Radioactivity was located predominantly in intermediate DNA during the first 10 min. After a short delay (extrapolated back to 3 min), label accumulated linearly in finished molecules. Figure 4B also shows that about 50% of the pulse label could be chased from intermediate DNA into completed viral molecules after adding a 1,000-fold excess of unlabeled thymidine at 10 min. The chase was effective within 10 min (Fig. 4B, inset). The kinetics of labeling
and the chase confirm a precursor-product relationship. Adenovirus DNA is replicated in 17 min, the time required to label intermediate DNA and completed molecules equally (i.e., the intersection in Fig. 4B). Intermediate DNA and completed viral molecules from each time point were also analyzed by velocity sedimentation in alkaline sucrose gradients (Fig. 5A through I). In agreement with Horwitz (9), no single strands longer than unit length (34S) were ever detected. After 1 min of labeling, strands of intermediate DNA (Fig. 5A) sedimented in the region of lOS (1,750 nucleotides) to 14S (3,500 nucleotides) with faster sedimenting shoulders at 19S (9,000 nucleotides) and 26S (17,500 nucleotides). Chains as short as 400 nucleotides could also be detected. Vlak et al. (33) and E. Winnacker (personal communication) have also found 9 to 11S strands as intermediates in adenovirus replication. Since the lOS chain corresponds to 'ho of an adenovirus strand, I have defined this unit as a "faceful" (i.e., equivalent to one of the 20 faces of the icosahedral virus particle). The signifi-
VOL. 16, 1975
REPLICATING ADENOVIRUS DNA
cance of facefuls will be discussed below. After 2 (Fig. 5B) or 3 min (Fig. 5C) of labeling, strands of intermediate DNA sedimented at 14, 18, 23, and 26S, which correspond to 2, 4, 8, and 10 facefuls. Longer strands of 30 to 32S (16 to 18 facefuls) appear by 4 (Fig. 5D) and 5 min (Fig. 5E). The size of nascent chains reached an equilibrium distribution of 26S within 10 min (Fig. 5F). Unit-length single strands were also present at this time. Further experiments to size nascent strands by agarose gel electrophoresis are in progress. At all times, completed molecules sedimented primarily as unit-length single strands. Some newly finished molecules evidently have transient single-strand interruptions. Finished molecules isolated after 10 (Fig. 5G) or 15 min (Fig. 5H) of labeling contain shorter fragments sedimenting at 18, 23, 26, and 31S (i.e., 1/4, 1/3, 1/2, and 3/4 fractional lengths). After a chase with unlabeled thymidine for 80 min (Fig. 5I), all strands were unit length. This also excludes
21
the possiblity that ethidium bromide introduced strand breaks during the separation of intermediate DNA from finished molecules. The center of mass of each sucrose gradient profile of intermediate DNA was plotted in Fig. 5J as a function of labeling time. Interestingly, the graph extrapolated to a molecular weight of 5.8 x 10' (or 1,750 nucleotides) for an infinitely short pulse. The initial rate of chain elongation was 0.7 x 106 daltons/min. The time to complete unit-length single strands was calculated to be 16 min, in close agreement with the estimate in Fig. 4B. Properties of intermediate DNA. Either single-stranded DNA or RNA could increase the buoyant density of intermediate DNA. Pettersson (14) showed that the density difference between replicating and mature adenovirus DNA was eliminated after digestion with the single-strand-specific nuclease from Neurospora crassa, but not after digestion with RNase. The following experiments confirm
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0 10 l0 20 30 20 30 l0 20 30 FRACTIONS FRACTIONS FRACTIONS FiG. 3. Characterization of intermediate DNA and completed viral DNA. A sample from the culture described in Fig. 2 was removed after labeling for 15 min. (A) Equilibrium centrifugation of extracted DNA in an ethidium bromide-cesium chloride density gradient. Fractions labeled b and c were pooled separately, extracted with isopropanol, and dialyzed. ["4C]HeLa DNA (not shown) co-banded with the light band (c). (B) Rebanding of fraction b and ["4CJHeLa DNA (not shown) in a neutral CsCI density gradient (initial p 1.710). Fractions labeled e were pooled and dialyzed. The vertical dashed lines represent, from left to right: intermediate DNA, adenovirus DNA, and HeLa DNA. (C) Rebanding of fraction c and ["4ClHeLa (not shown) in a neutral CsCI density gradient. Fractions labeled f were pooled and dialyzed. (D) Rebanding of a mixture of b and c in a neutral CsCI density gradient. Closed circles represent marker ["4C]HeLa DNA. (E) Neutral sucrose gradient velocity sedimentation of fraction e. Sucrose gradients (containing 1.0 M NaCI, 0.005 M EDTA, and 0.01 M Tris, pH 8) were centrifuged for 7 h at 25,000 rpm. Fractions labeled h and i were pooled separately and dialyzed. (F) Neutral sucrose gradient velocity sedimentation offraction f. The fraction labeled j was retained and dialyzed. (G) Neutral sucrose gradient velocity sedimentation of a mixture of e and f. The arrow marks the position of 42S. (H) Rebanding of fraction h in a neutral CsCI density gradient. Symbols: *, [CJC]HeLa DNA. The vertical dashed lines represent, from left to right: intermediate DNA, adenovirus DNA, and HeLa DNA. (1) Rebanding offraction i and ["4C JHeLa DNA (not shown) in a neutral CsCI density gradient. (J) Rebanding of fraction j and ["C JHeLa DNA (not shown) in a neutral CsCI density gradient. 0
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FIG. 4. Kinetics of labeling intermediate DNA and finished adenovirus DNA. Infected cells were concentrated to 4 x 106 cells/ml and labeled with [9H]thymidine (10 tiCi/ml at 10 Ci/mmol). At 10 min the incorporation of [3H]thymidine was quenched by adding 10-4 M unlabeled thymidine. Samples of 4 x 106 cells were removed at the indicated times, extracted, and centrifuged to equilibrium in cesium chloride-ethidium bromide gradients. (A) Profiles of cesium chloride-ethidium bromide density gradients. The peak of [14C]HeLa DNA (not shown) is indicated by the arrow. The [9H]thymidine incorporated into adenovirus DNA was normalized to the recovery of HeLa DNA in each gradient (45,000 counts/min of 14C). Symbols: 0, 1 min; 0, 3 min; A, 5 min; 0, 10 min. (B) Radioactivity in the peak fractions of the cesium chloride-ethidium bromide gradients was normalized for recovery of HeLa DNA in each gradient and plotted as a function of the time of labeling. The chase with 10-4 M thymidine was started 10 min after adding the [3H]thymidine. Symbols: 0, 3H in intermediate DNA; 0, 3H in completed adenovirus DNA. (Insert) Total incorporation of. ['H]thymidine in each gradient as a function of time. The dashed line indicates the initial rate of incorporation. The arrow indicates the beginning of the chase.
those observations. Purified intermediate DNA, labeled for 5 min with ['H ]thymidine, was mixed with [14C ]adenovirus DNA and digested with pancreatic RNase or the single-strandspecific Sl endonuclease from A. oryzae (1, 28). Intermediate DNA had a buoyant density of 1.725 (Fig. 6A). After treatment with Si endonuclease (Fig. 6B), intermediate DNA banded at the density of marker DNA, although less than 1% of 3H and '4C labels were solubilized. Under the same conditions, more than 95% of heat-denatured viral DNA could be digested. Figure 6C shows that RNase digestion in buffer containing 0.2 M NaCl had no effect on the density of intermediate DNA. RNase hydrolyzed better than 98% of ['H ]HeLa rRNA under the same conditions. These experiments strongly suggest that the increased buoyant density of intermediate DNA is due to substan-
tial single strands of parental DNA. In this regard, replicating adenovirus chromosomes appear in electron micrographs as branched and unbranched linear molecules with extensive single-stranded regions (8, 23, 26, 29). Thus, adenovirus might replicate by displacement synthesis, a mechanism proposed for mitochondrial DNA replication (3, 16, 17). In spite of the above conclusions, hydrogenbonded RNA may play a role in maintaining the structure of intermediate DNA. Figure 6D demonstrates that RNase disrupted intermediate DNA in 0.02 M NaCl, conditions where RNase attacks RNA in RNA-DNA hybrids. In three experiments an average of 9% (range, 6 to 12%) of the 3H label banded at the position of marker DNA. The rest of the 3H activity floated at the meniscus of the CsCl gradient (the total recovery of 3H was better than 70%). An interpreta-
VOL. 16, 1975
REPLICATING ADENOVIRUS DNA
tion is that nascent strands in intermediate DNA are released as a low density complex. The complex must be insensitive to detergents, Pronase, and concentrated salt solutions. Pettersson (14) also reported that RNase digestion in buffer lacking salt reduces the buoyant density of replicating adenovirus DNA, but the magnitude of the change was less than reported here. Furthermore, intermediate DNA can be labeled with radioactive uridine (7; G. D. Pearson, unpublished data). The sedimentation rate of intermediate DNA is markedly altered by changes in ionic strength. Figure 7A demonstrates that intermediate DNA formed a fast sedimenting aggregate (greater than 100S) when the ionic strength was lowered to 0.01 M NaCl. At salt concentrations greater than 0.1 M NaCl, intermediate DNA sedimented as a broad zone with peaks at 47 and 31S (Fig. 7B; compare with Fig. 2B and
23
3E). The sedimentation rate of marker adenovirus DNA was not affected within the range of ionic strengths used in these experiments. It is difficult to attribute this behavior to singlestranded regions in intermediate DNA. Single DNA chains assume an extended conformation in low salt due to electrostatic repulsion by negatively charged phosphates. The sedimentation rate decreases as a consequence (22). Although the basis for the aggregation is not known, it has been used to separate finished molecules from replicating molecules to construct a replication map of adenovirus (G. D. Pearson, manuscript in preparation) by the method of Danna and Nathans (4). DISCUSSION Nascent DNA contained in the adenovirus replication complex, called intermediate DNA, was shown to differ physically from mature viral
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10 l0 20 30 l0 20 30 FRACTIONS FRACTIONS FRACTIONS FIG. 5. Alkaline sucrose gradient velocity sedimentation of intermediate DNA and completed adenovirus DNA isolated from the cesium chloride-ethidium bromide gradients described in Fig. 4. Peak fractions were pooled separately, extracted with isopropanol, and dialyzed. The fractions were then denatured by adjusting to 0.2 M NaOH and centrifuged for 7 h at 25,000 rpm in alkaline sucrose gradients (containing 1.0 M Na+). At least 2,000 counts/min of 3H were in each gradient. (A) Intermediate DNA, 1-min pulse; (B) intermediate DNA, 2-min pulse; (C) intermediate DNA, 3-min pulse; (D) intermediate DNA, 4-min pulse; (E) intermediate DNA, 5-min pulse; (F) intermediate DNA, 10-min pulse; (G) completed viral DNA, 10-min pulse; (H) completed viral DNA, 10-min pulse plus 5-min chase; (I) completed viral DNA, 10-min pulse plus 80-min chase; (J) plot of the center of mass of intermediate DNA in each gradient as a function of the labeling time. The center of mass was calculated from the relationship . ,f1CIC,, where f, fraction number and Cl counts per minute in that fraction. This was converted to single-stranded molecular weight (22). 0
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FRACTIONS F I G. 6. Digestion of intermediate DNA with Sl endonuclease or ribonuclease. ['H]intermediate DNA was purified as described in Fig. 2, For Sl endonuclease, the reaction contained per 0.3 ml: 25 Ml of ['HJintermediate DNA; 10 MI of ['4C]adenovirus DNA; 7.5 Mg of heat-denatured calf thymus DNA; 25 MI of enzyme in 0.07 M NaCl, 0.03 M sodium citrate, 0.001 M ZnCI2, pH 4.5. The mixture was incubated for 1 h at 50 C, stopped by adding 0.5% sarkosyl, and centrifuged in CsCI gradients containing 0.5% sarkosyl. RNase was first heated to 80 C for 10 min. Reactions in high salt contained per 1.0 ml: 25 Al of [(H]intermediate DNA, 10 Al of ['4C]adenovirus DNA, and 100 Mg ofpancreatic RNase in 0.2 MNaCI, 0.01 M EDTA, 0.01 M Tris, pH8. After I h at 37 C, the reactions were stopped by adding 0.5% sarkosyl and centrifuged as above. Reactions in low salt were identical except that the buffer contained: 0.02 M NaCI, 0.01 M EDTA, 0.01 M Tris, pH 8. Symbols: 0, [3H]intermediate DNA; 0, ['4C]adenovirus DNA. (A) No Sl endonuclease (control). The controls in high and low salt without RNase gave similar profiles (not shown); (B) with Sl endonuclease; (C) with pancreatic RNase in 0.2 M NaCI; (D) with pancreatic RNase in 0.02 M NaCI.
molecules. For example, intermediate DNA had an increased buoyant density (1.725 compared to 1.715) and a greater sedimentation coefficient (up to 100S compared to 31S). Since treatment with the single-strand-specific Si endonuclease, but not with RNase, lowered the buoyant density of intermediate DNA without loss of the pulse label, a substantial fraction of the parental strands must be single stranded (also see 14). Branched and unbranched linear molecules containing extensive single-stranded regions have been visualized in the electron microscope (8, 23, 26, 29). These properties agree with the properties of replicating adenovirus DNA reported by other laboratories (2, 8, 23-26, 29, 32). An improved method for isolating intermediate DNA has been developed. Ethidium bromidecesium chloride density gradients provide increased separation between intermediate DNA (1.605) and mature viral DNA (1.585). Experiments reported here carefully document the identities of these bands. When labeled for 1 min or less, nascent strands from intermediate DNA sedimented
primarily at lOS, corresponding to a chain 1,750 nucleotides long. No strands longer than an intact viral strand were ever detected. Vlak et al. (33) and E. Winnacker (personal communication) have shown that 9 to 11S nascent chains are complementary to both viral strands throughout the entire genome. Remarkably, other nascent strands at this and longer times of labeling were integral multiples of 1,750 nucleotides. This replication unit has been defined as a faceful, since it is exactly '/2 of a finished adenovirus strand. Recent experiments suggest a relationship between replication units and the nucleoprotein structure of the virus chromosome. Double-stranded equivalents of the faceful (i.e., 1,750 base pairs) appear as the initial product when disrupted adenovirus particles are digested with staphylococcal nuclease. Longer digestion times yield homogeneous fragments of about 150 base pairs. We postulate that a regular arrangement of core proteins protect discrete regions of viral DNA from nuclease attack. A complete model with supporting evidence will be published elsewhere (J.
REPLICATING ADENOVIRUS DNA
VOL. 16, 1975
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FRACTIONS FIG. 7. Effect of ionic strength on the sedimentation rate of intermediate DNA. ['H]intermediate DNA was mixed with ["ICfadenovirus DNA and sedimented for 5 h at 25,000 rpm in neutral sucrose gradients containing 0.01 M NaCI, 0.Ou M EDTA, and 0.01 M Tris, pH8 (A), or U.1 M NaCI, 0.001 M EDTA, and 0.01 M Tris, pH 8 (B). The prorfue o0 intermediate DNA in gradients containing 1.0 M NaCI, 0.001 M EDTA, and 0.01 M Tris, pH 8, resembled the profile in (B). Symbols: 0, ['H]intermediate DNA; *, [(1C adenovirus DNA.
Corden, H. M. Engelking, and G. Pearson, manuscript in preparation). The average time required for adenovirus replication was calculated to be 16 to 17' min, corresponding to a rate of 0.7 x 106 daltons/min for chain elongation. This rate compares favorably with 0.5 x 106 daltons/min for chicken embryo lethal orphan virus, an avian adenovirus (2), 0.6 x 106 daltons/min for simian virus 40 (12), and 0.8 x 106 daltons/min for the L strand of mitochondrial DNA (3). Pearson and Hanawalt (13) previously overestimated the replication rate because excessive shear was used to isolate the replication complex. Although newly finished molecules sedimented at 31S and had a density of 1.715, they still contained single-strand interruptions. Mature chromosomes do not. Complete joining of daughter strands required at least an additional 15 to 20 min (unpublished observations). Unjoined strands were approximately 0.25, 0.5, and 0.75 fractional lengths. Interestingly, an adenovirus replication map (Pearson, manuscript in preparation), constructed by the method of Danna and Nathans (4), locates an origin in Eco RI fragment F or in Eco RI fragment B very near to fragment F and at least
one other origin within the Eco RI fragment A in a region defined by the Hpa I fragment F (E. Winnacker, personal communication). The sizes (15) and order (11) of the Eco RI endonuclease fragments of adenovirus DNA have been reported. Thus, two replicative origins are positioned about 25% from either end of the chromosome. Horwitz (10) also has demonstrated origins in both halves of the adenovirus genome using a similar approach. Unbranched molecules with many single-stranded gaps have been visualized by electron microscopy (8, 26, 29). It is tempting to speculate that unjoined strands are sealed at the junctions between origins and termini. ACKNOWLEDGMENTS This research was supported by grants (NP-67, NP-67A) from the American Cancer Society and a Biomedical Sciences Support Grant (5 S05 RR07079-06) administered through the Oregon State University Research Council. Part of this work was done as a Dernham Junior Fellow (J-126) of the California Division of the American Cancer Society. I thank Philip Hanawalt, John Kiger, Ernst Winnacker, David Dressler, and John Wolfson for many valuable suggestions and discussions. I also thank John Sussenbach for a copy of his paper prior to publication. Gail Foster provided devoted technical assistance.
26
PEARSON LITERATURE CITED
1. Ando, T. 1966. A nuclease specific for heat-denatured DNA isolated from a product of Aspergillus oryzae. Biochem. Biophys. Acta 114:158-168. 2. Bellett, 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. Berk, A. J., and D. A. Clayton. 1974. Mechanism of mitochondrial DNA replication in mouse L-cells; asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence. J. Mol. Biol. 86:801-824. 4. Danna, K. J., and D. Nathans. 1972. Bidirectional replication of simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 69:3097-3100. 5. Doerfler, W. 1969. Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology 38:587-606. 6. Doerfler, W., U. Lundholm, and M. Hirsch-Kauffmann. 1972. Intracellular forms of adenovirus deoxyribonucleic acid. I. Evidence for a deoxyribonucleic acid protein complex in baby hamster kidney cells infected with adenovirus type 12. J. Virol. 9:297-308. 7. Doerfler, W., U. Lundholm, U. Rensing, and L. Philipson. 1973. Intracellular forms of adenovirus DNA. II. Isolation in dye-buoyant density gradients of a DNA-RNA complex from KB cells infected with adenovirus type 2. J. Virol. 12:793-807. 8. Ellens, D. J., J. S. Sussenbach, and H. S. Jansz. 1974. Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA. Virology 61:427-442. 9. Horwitz, M. S. 1971. Intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid. J. Virol. 8:675-683. 10. Horwitz, M. S. 1974. Location of the origin of DNA replication in adenovirus type 2. J. Virol. 13:1046-1054. 11. Mulder, C., P. A. Sharp, H. Delius, and U. Pettersson. 1974. Specific fragmentation of DNA of adenovirus serotypes 3, 5, 7, and 12, and adeno-simian virus 40 hybrid virus Ad2+ND1 by restriction endonuclease R-EcoRI. J. Virol. 14:68-77. 12. Nathans, D., and K. J. Danna. 1972. Specific origin in SV40 DNA replication. Nature (London) New Biol. 236:200-202. 13. Pearson, G. D., and P. C. Hanawalt. 1971. Isolation of, DNA replication complexes from uninfected and adenovirus-infected HeLa cells. J. Mol. Biol. 62:65-80. 14. Pettersson, U. 1973. Some unusual properties of replicating adenovirus type 2 DNA. J. Mol. Biol. 81:521-527. 15. Pettersson, U., C. Mulder, H. Delius, and P. Sharp. 1973. Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R -RI. Proc. Natl. Acad. Sci. U.S.A. 70:200-204. 16. Robberson, D. L., and D. A. Clayton. 1972. Replication of mitochondrial DNA in mouse L cells and their thymidine kinase derivatives: displacement replication on a covalently-closed circular template. Proc. Natl. Acad.
J. VIROL. Sci. U.S.A. 69:3810-3814. 17. Robberson, D. L., and D. A. Clayton. 1973. Pulse-labeled components in the replication of mitochondrial deoxyribonucleic acid. J. Biol. Chem. 248:4512-4514. 18. Robinson, A. J., H. B. Younghusband, and A. J. D. Bellett. 1973. A circular DNA-protein complex from adenoviruses. Virology 56:54-69. 19. Shiroki, K., and H. Shimojo. 1974. Analysis of adenovirus 12 temperature-sensitive mutants defective in viral DNA replication. Virology 61:474-485. 20. Shiroki, K., H. Shimojo, and K. Yamaguchi. 1974. The viral DNA replication complex of adenovirus 12. Virology 60:192-199. 21. Simmons, T., P. Heywood, and L. D. Hodge. 1974. Intranuclear site of replication of adenovirus DNA. J. Mol. Biol. 89:423-433. 22. Studier, F. W. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390. 23. Sussenbach, J. S., D. J. Ellens, and H. S. Jansz. 1973. Studies on the mechanism of replication of adenovirus DNA. J. Virol. 12:1131-1138. 24. Sussenbach, J. S., and P. C. van der Vliet. 1972. Viral DNA synthesis in isolated nuclei from adenovirusinfected KB cells. FEBS Lett. 21:7-10. 25. Sussenbach, J. S., and P. C. van der Vliet. 1973. Studies on the mechanism of replication of adenovirus DNA. I. The effect of hydroxyurea. Virology 54:299-303. 26. Sussenbach, J. S., P. C. van der Vliet, D. J. Ellens, and H. S. Jansz. 1972. Linear intermediates in the replication of adenovirus DNA. Nature (London) New Biol. 239:47-49. 27. Suzuki, E., and H. Shimojo. 1974. Temperature-sensitive formation of the DNA replication complex in adenovirus 31-infected cells. J. Virol. 13:538-540. 28. Sutton, W. D. 1971. A crude nuclease preparation suitable for use in DNA reassociation experiments. Biochim. Biophys. Acta 240:522-531. 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 A. J. Levine. 1972. DNA-binding proteins specific for cells infected by adenovirus. Nature (London) New Biol. 246:170-174. 31. van der Vliet, P. C., A. J. Levine, M. J. Ensinger, and H. S. Ginsberg. 1975. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Virol. 15:348-354. 32. 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. 33. Vlak, J. M., T. H. Rozijn, and J. S. Sussenbach. 1975. Studies on the mechanism of replication of adenovirus DNA. IV. Discontinuous DNA chain propagation. Virology 63:168-175. 34. Yamashita, T., and M. Green. 1974. Adenovirus DNA replication. I. Requirement for protein synthesis and isolation of nuclear membrane fractions containing newly synthesized viral DNA and proteins. J. Virol. 14:412-420.
