1800 base pair fragments ('o of the adenovirus chromosome). This result is discussed in terms of a higher order folding of viral DNA within the virus particle.
Proc. Nat. Acad. Sci. USA Vol. 73, No. 2, pp. 401-404, February 1976
Biochemistry
Chromatin-like organization of the adenovirus chromosome (staphylococcal nuclease/nucleoprotein structure/core polypeptides/virus assembly)
JEFF CORDEN, H. MARK ENGELKING, AND GEORGE D. PEARSON* Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oreg. 97331
Communicated by Harold J. Evans, November 24, 1975
mentation in a sucrose gradient. All preparations were adjusted to 1 mM CaCl2 and pH 7.5. Isolation of Nuclei. Cells were centrifuged at 500 X g for 5 min and resuspended at a concentration of 107 cells per ml in buffer containing 0.3 M sucrose, 10 mM Tris-cacodylate, 3 mM CaCl2, 0.5% Nonidet P-40, pH 7.8. Cells were lysed at 20 by repeatedly drawing (about 20 times) the suspension into a pasteur pipet. After 5 min, the nuclei were pelleted at 500 X g for 10 min and resuspended at 107 nuclei per ml in 0.3 M sucrose, 10 mM Tris-cacodylate, 1 mM CaCl2, pH 7.8. Nuclease Digestion. Staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase; EC 3.1.4.7) was purchased from Worthington Biochemical Corp. Reaction mixtures were incubated at 370 and reactions were terminated by adjusting the solutions to 10 mM EDTA, 1 mg of Pronase per ml, 2% Sarkosyl, and 10% glycerol. After 1 hr at 370, samples were applied directly to gels. One unit of nuclease corresponds to a change of 1 A260 unit at 37°. Gel Electrophoresis. Agarose (Sigma) gels containing 0.5 ,ug of ethidium bromide per ml were cast in 6 mm X 30 cm Plexiglas tubes. The running buffer, E buffer (18), contained 0.4 ,tg of ethidium bromide per ml. Electrophoresis was at 5 mA per gel for the indicated times. Gels were cut into 0.2 inch (0.5 cm) slices. Each slice was placed with 0.25 ml of H20 into 1 dram vials, autoclaved, and mixed with 2.5 ml of Aquasol. The radioactivity was determined by scintillation counting.
ABSTRACT Staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase; EC 3.1.4.7) cleaved DNA within disrupted adenovirus particles into a regular series of fragments with a repeat unit of 200 base pairs. Since this pattern did not appear when DNA alone was digested, we postulate that the orderly arrangement of core polypeptides protects discrete regions of DNA from nuclease attack. The 23 X 106 dalton adenovirus DNA molecule can accommodate 180 units of roughly 200 base pairs. Based on the stoichiometry of core polypeptides, we calculate that each repeat unit contains six copies of polypeptide VII and a single copy of polypeptide V. This model is based on proposals for the structure of eukaryotic chromatin. Very brief nuclease digestion generated 1800 base pair fragments ('o of the adenovirus chromosome). This result is discussed in terms of a higher order folding of viral DNA within the virus particle. Although the capsid architecture of the adenovirus particle is known in great detail, no fundamental understanding of the internal structure exists. Recent proposals for the structure of eukaryotic chromatin (1, 2) suggest a powerful model for the organization of the adenovirus core. The DNA in chromatin is wound around (3) specific histone complexes (4, 5) to form repeating subunits (6-8). The chromosomes of at least two animal viruses, polyoma and simian virus 40, are also condensed into subunits (9-11) within virus particles by complexing with cellular histones (12, 13). The adenovirus core does not contain histones, but we show in this paper that the core has a chromatin-like design. MATERIALS AND METHODS Cell Culture and Synchronization. HeLa S3 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 hr to 2 mM thymidine separated by a period of 12 hr (14). Adenovirus Infection. Inocula for all experiments consisted of type 2 adenovirus purified in CsCl density gradients according to Doerfler (15). Cells were infected with 104 particles per cell as previously described (14). Virus labeled with [methyl-3H]thymidine (New England Nuclear) had a specific activity of 5 X 104 cpm/,ug of viral DNA. Disruption of Adenovirus. [3H]Thymidine-labeled virus particles were disrupted by (a) heating to 560 for 90 sec in 5 mM Tris buffer at pH 7.5 (16), (b) dialysis against 5 mM Tris-maleate buffer at pH 6.4 as described by Prage et al. (17), or (c) treatment with 10% pyridine in 5 mM Tris buffer at pH 8.1 (17). The first two methods yielded "cornerless" particles which were not separated from free capsomers. Pyridine treatment stripped off the entire capsid. The resulting cores were further purified from capsomers by sedi-
RESULTS
Nuclease digestion of disrupted adenovirus particles We have used staphylococcal nuclease (EC 3.1.4.7) to probe the structure of the adenovirus core. Adenovirus particles dialyzed against 5 mM Tris-maleate buffer at pH 6.4 lose pentons and neighboring hexons (17). The rest of the capsid, a shell of 180 hexons, still surrounds the core. Staphylococcal nuclease penetrates such "cornerless" particles and attacks viral DNA in the core. Fig. 1 shows the kinetics of nuclease digestion. The initial rate of the reaction was proportional to the nuclease concentration. However, only 55% of the viral DNA was acid soluble at either concentration of nuclease. All of the remaining DNA had been cut into pieces 200 base pairs (bp) or smaller (see below). The hydrolysis of proteinfree adenovirus DNA did not stop at this well-defined limit. Nuclease-resistant viral DNA was sized by electrophoresis on 1.5% agarose gels containing ethidium bromide. Eco RI fragments of type 2 adenovirus DNA (19) and Hae III fragments of PM2 DNA (R. T. Kovacic, manuscript in preparation) were used to calibrate the gels. These overlapping sets of fragments span a molecular weight range from greater than 20,000 bp to 55 bp. The inverse relationship between the logarithm of the molecular weight and the distance migrated is linear on these gels from 2600 bp to 200 bp. Fig.
Abbreviation: bp, base pairs. * To whom reprint requests should be sent. 401
402
Proc. Nat. Acad. Sci. USA 73 (1976)
Biochemistry: Corden et al.
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FIG. 1. Kinetics of nuclease digestion. [3H]Adenovirus disrupted by dialysis against 5 mM Tris-maleate buffer at pH 6.4 was digested at a concentration of 8 A260) units/ml (1 A26rs unit = 1012 particles) with staphylococcal nuclease. Duplicate samples were removed at the indicated times. One sample was immediately oxidized in a Packard model 306 Sample Oxidizer. The maximum radioactivity was 1000 I 100 cpm. The other sample was precipitated with 7% trichloroacetic acid and a portion of the supernatant oxidized. The percent acid-soluble nucleotides was calculated. The dashed lines indicate the initial rates of reaction. 0, 1 unit of nuclease per ml; 0, 2 units of nuclease per ml.
2A shows that about 10% of the resistant DNA had been cleaved into 1800 bp fragments (1ko of the adenovirus chromosome) early in the reaction. The bulk of the DNA migrated as intact molecules (actually, fragments larger than 6000 bp are not resolved on these gels). The small band at 330 bp never appeared in any other experiment and is likely an artifact. Extensive digestion converted over 50% of the nuclease-resistant DNA into 200 bp or smaller fragments (Fig. 2B). These fragments were estimated to be 195 bp and 85 bp, respectively, on 3.5% acrylamide gels (not shown). The rest of the DNA in Fig. 2B was essentially intact, but in other experiments there was no high-molecular-weight DNA late in the reaction. Staphylococcal nuclease did not attack DNA in undamaged virus particles. Furthermore, discrete fragments were never produced when protein-free adenovirus DNA was digested. The specific cleavage of viral DNA in the core does not depend on the method of disrupting adenovirus. "Cornerless" particles can also be prepared by heating adenovirus at 560 (16). Nuclease digestion of heat-shocked particles again generated 1800 bp fragments early in the reaction and 200 bp or smaller fragments later (not shown). The adenovirus core can be isolated after the entire capsid is removed by 10% pyridine (17). In addition to viral DNA, pyridine cores contain essentially two polypeptides called V and VII (20, 21). The core polypeptides are virus gene products and have been mapped on the left half of the chromosome (22). Fig. 3A shows that staphylococcal nuclease cut DNA in pyridine cores into 200 bp and smaller fragments. No band appeared at 1800 bp even though the reaction was only 1/o as vigorous as the digestion in Fig. 2A. The significance of this observation will be discussed below. Neverthe-
less, some DNA as large as 600 bp was present. Exhaustive digestion (180 times as long as in Fig. 3A and equivalent to the reaction in Fig. 2B) converted all nuclease-resistant DNA into 200 bp fragments or less (Fig. 3B). Since adenovirus cores do not contain cellular histones, the orderly binding of basic core polypeptides must produce the discrete pattern of nuclease-resistant DNA fragments. Nuclease digestion of intranuclear adenovirus DNA Synchronized cells infected for 13 hr were pulse-labeled for 1 hr with [3H]thymidine and then chased for 1 hr with unla-
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