Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure

Varshavsky, Alexander; Nedospasov, Sergei A.; Schmatchenko, Vadim; Bakayev, Valery V.; Chumackov, P.M.; Georgiev, G.P.

doi:10.1093/nar/4.10.3303

Cited by 65 publications

(37 citation statements)

References 30 publications

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“…Alternatively, the rigidity and constraints provided by a continuous DNA chain could be important in order to modulate interactions of histones Hi and H5 in such a way that, for instance, nucleosomal chains fold into solenoids instead of superbeads. Nevertheless, we think this is unlikely, because in the presence of moderate salt concentrations (about 0.1 M NaCl) the 24 nucleosomes of the simian virus 40 minichromosome are also densely packed in a spherical particle with a diameter of about 300 A (31,32). These native compact minichromosomes are practically indistinguishable from the globular particles we have described.…”

Section: Methodsmentioning

confidence: 77%

Aggregation of small oligonucleosomal chains into 300-A globular particles.

Jorcano¹,

Meyer²,

Day³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Chicken erythrocyte oligonucleosomes (trimers to about 20-mers) are able to interact with each other through the very Iysine-rich histones (HI and H5) and form heterogeneous globular particles with a mean diameter of about 300 A. These particles assemble spontaneously during micrococcal nuclease digestion of chromatin in the presence of 30 mM NaCl and contain approximately 25 nucleosomes. They are sensitive to ionic strength and unfold at lower salt concentrations but can be reconstituted by restoring the initial salt concentration. Even at 30 mM NaCl, the particles remain dynamic structures, being in equilibrium with their oligonucleosomal components as revealed by the fact that particle stability depends on the concentration of oligonucleosomes. Almost all DNA in nuclei of eukaryotic cells is complexed with histones to form linear arrays of a basic subunit called the nucleosome (1, 2). The nucleosomal chain, which has a diameter of approximately 110 A, is folded in a still unknown fashion into a thicker chromosomal fiber (250-300 A in diameter) (3-5). Two molecular models have been proposed to explain the structure of the chromosomal fiber. In the first, nucleosomes are continuously aligned in a long helix (solenoid) (6-9), whereas in the second nucleosomes are somehow clustered in discrete globular particles (superbeads) (10-13). In any case, many experimental results demonstrate that the very lysine-rich histones (histones of the HI class and histone H5) organize the supranucleosomal packaging through interactions with nucleosomes and probably also with themselves (6,9,14,15).Binding of very lysine-rich histones to DNA is not sufficient to induce chromatin condensation into a thick fiber. At lowNaCl concentrations chromatin appears as a linear chain of nucleosomes, although very lysine-rich histones remain bound to it (6,9,14). Increasing the NaCl concentration leads to a folding of the nucleosome chain into the 250-to 300-A fiber (6,9,14). This transition occurs in a rather narrow salt range (between 20 and 40 mM NaCl), which coincides with the range in which very lysine-rich histones change their binding mode to DNA from a noncooperative to a cooperative interaction (14,16). This suggests that interactions between these histones are important for the maintenance of the 250-to 300-A fiber configuration. In this communication we describe experiments which show that this is indeed the case. Workinq at 30 mM NaCI, an ionic strength at which the 250-to 300-A fiber is already unstable, we found that small oligonucleosomes aggregate into compact globular structures with a diameter (about 300A) very similar to the thickness of the native chromosomal fiber.The folded and unfolded states of these structures are found to be in equilibrium under these rather destabilizing conditions.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6443The formation ...

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Section: Methodsmentioning

confidence: 77%

Aggregation of small oligonucleosomal chains into 300-A globular particles.

Jorcano¹,

Meyer²,

Day³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…SV40 is known to be sensitive to treatment with chelating agents and reducing agents (2, 37), which render the viral DNA sensitive to DNase I treatment (61). Following treatment with 5 mM EGTA and 5 mM DTT, the wild-type and Vp1-only particles were equally sensitive to DNase I digestion (Fig.…”

Section: Resultsmentioning

confidence: 98%

Minor Capsid Proteins of Simian Virus 40 Are Dispensable for Nucleocapsid Assembly and Cell Entry but Are Required for Nuclear Entry of the Viral Genome

Nakanishi

Itoh

et al. 2007

J Virol

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We investigated the roles of simian virus 40 capsid proteins in the viral life cycle by analyzing point mutants in Vp1 and Vp2/3, as well as a deletion mutant lacking the Vp2/3 coding sequence. The Vp1 mutants (V243E and L245E) and the Vp2/3 mutants (F157E-I158E and P164R-G165E-G166R) were previously shown to be defective in Vp1-Vp2/3 interaction and to be noninfectious or poorly infectious, respectively. Here, we show that all these point mutants form stable particles following DNA transfection into cells. The Vp2/3-mutant particles contained very low levels of Vp2/3, whereas the Vp1 mutant particles contained no detectable Vp2/3. As expected, the deletion mutant also formed particles that were noninfectious. We further characterized the two Vp1 point mutants and the deletion mutant. All three mutant particles comprised Vp1 and histone-associated viral DNA, and all were able to enter cells. However, the mutant complexes failed to associate with host importins (owing to the loss of the Vp2/3 nuclear localization signal), and the mutant viral DNAs prematurely dissociated from the Vp1s, suggesting that the nucleocapsids did not enter the nucleus. Consistently, all three mutant particles failed to express large T antigen. Together, our results demonstrate unequivocally that Vp2/3 is dispensable for the formation of nucleocapsids. Further, the nucleocapsids' ability to enter cells implies that Vp1 contains the major determinants for cell attachment and entry. We propose that the major role of Vp2/3 in infectivity is to mediate the nuclear entry of viral DNA.

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“…The relaxation phenomenon is more apparent at higher ionic strengths (20 and 75 mM NaCl), in which control nucleosomes undergo a gradual condensation toward the 250-to 300-A fibers and in which poly(ADP-ribosyl)ated nucleosomes maintain an open structure. The transition of the chromatin structure from a zigzag to a compact form under high ionic strength was described to be dependent upon the presence of histone H1 (21,31) ribose) showed that histone H1 was the major poly(ADP-ribosyl)ated histone. The possibility that poly(ADP-ribosyl)ation of histone H1 detaches these molecules from the chromatin is unlikely because under our experimental conditions no histone H1 was detected on top of the sucrose gradient onto which a poly(ADP-ribosyl)ated nucleosome mixture had been loaded (see Figs.…”

Section: Discussionmentioning

confidence: 99%

Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure.

Poirier¹,

Murcia²,

Jongstra‐Bilen³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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When rat pancreatic polynucleosomes were poly(ADP-ribosyl)ated with purified calfthymus poly(ADP-ribose) polymerase and examined by electron microscopy, a relaxation of their native zigzag structure was observed. At high ionic strengths control nucleosomes condensed into 250-A-thick fibers, but poly(ADP-ribosyl)ated polynucleosomes did not; they showed a close resemblance to chromatin depleted of histone HI. The relaxed state of poly(ADP-ribosyl)ated polynucleosomes was also confirmed by sedimentation velocity analysis. Histone HI was found to be the major histone acceptor of poly(ADP-ribose). Poly(ADP-ribose) linked to histone HI did not seem to cause its dissociation from the chromatin, but it impaired significantly its effect on chromatin condensation.Post-translational modifications of histones-e.g., phosphorylation (1), acetylation (2, 3), and poly(ADP-ribosyl)ation (4,5) have been suggested as possible mechanisms to modulate the nucleosomal structure during DNA transcription and replication.Poly(ADP-ribose) polymerase was suggested to be preferentially located at internucleosomal regions of HeLa cell nucleosomes (6, 7); its specific activity was shown to be highest at selected folded regions of 8-10 nucleosomes in higher-ordered chromatin (8). In addition, the poly(ADP-ribosyl)ation of several chromatin components, including all the histones, was demonstrated (9, 10). All these findings reinforced the suggestions of involvement of poly(ADP-ribosyl)ation in DNA replication, DNA repair, and gene expression through alteration of the chromatin structure.In order to test this hypothesis, pancreatic polynucleosomes (20-40 nucleosomes) were poly(ADP-ribosyl)ated by using purified calf thymus poly(ADP-ribose) polymerase. Changes of nucleosome structure were examined by electron microscopy and by ultracentrifugation and the poly(ADP-ribosyl)ated histones were characterized.MATERIALS AND METHODS Preparation ofPolynucleosomes. Rat pancreatic polynucleosomes were isolated as described (11). Nuclei were digested with micrococcal nuclease (Sigma) at 30°C for 2 min, with 0.5 unit of nuclease per mg of DNA. The polynucleosomes (20-40 nucleosomes) were isolated on linear 5-29% sucrose gradients by centrifugation at 260,000 X g for 150 min in a Beckman SW 41 rotor and were characterized by electron microscopy and circular dichroism.Poly(ADP-ribosyl)ation of Polynucleosomes. Polynucleosomes were poly(ADP-ribosyl)ated by using purified calf thymus poly(ADP-ribose) polymerase at 25°C in an incubation medium of 500 utl containing 8 jig of DNA-independent enzyme (12,13) Electron Microscopy. Chromatin samples diluted to 0.5 ,g/ ml (expressed as DNA concentration) with a buffer containing 5 mM triethanolamine at pH 7.4, 0.2 mM EDTA, and the appropriate concentration of NaCl (see figure legends) were fixed in 0.1% glutaraldehyde (Balzers, Lichtenstein) for 1 hr at room temperature. Adsorption of the specimens onto positively charged carbon-coated grids (400 mesh) was performed as described (14). The specimens were stained w...

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Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure

Cited by 65 publications

References 30 publications

Aggregation of small oligonucleosomal chains into 300-A globular particles.

Aggregation of small oligonucleosomal chains into 300-A globular particles.

Minor Capsid Proteins of Simian Virus 40 Are Dispensable for Nucleocapsid Assembly and Cell Entry but Are Required for Nuclear Entry of the Viral Genome

Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure.

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