Topological properties of bovine papillomavirus type 1 (BPV-1) DNA in episomal nucleoprotein complexes: a model system for chromatin organization in higher eukaryotes.
Sedimentation analysis of isolated episomal bovine papillomavirus type 1 (BPV-1) nucleoprotein complexes in sucrose gradients and subsequent separation of the purified DNA in chloroquine gels revealed different classes of molecules, varying in their degree of superhelicity. Since torsionally stressed DNA favors the adoption of secondary structures, we employed the single-strand-specific S1 nuclease to detect such structural alterations in both naked DNA and native chromatin. Direct examination of nuclease digestion products in chloroquine gels showed that neither the naked DNA nor the BPV-1 nucleoprotein complexes in isolated nuclei were cleaved randomly by the enzyme. Instead, there was a strict dependence on nuclease susceptibility and the degree of supercoiling, strongly suggesting that the structural features detected by S1 nuclease are due to the occurrence of torsionally stressed viral chromatin. Mapping analysis using the indirect end-labeling method demonstrated an S1-nuclease cleavage site adjacent to 20 homopurine residues known to be hypersensitive to S1 attack. Furthermore, direct methylation experiments with viral chromatin in isolated nuclei indicated that only circular, covalently closed nucleoprotein complexes served as substrate, whereas linearized BPV-1 chromatin was not susceptible to exogenously added Hhal methylase. This observation raises the possibility that the modulation of topology in nucleosomally organized DNA might also play a role in eukaryotic DNA methylation.
Rösl
F
F
Institut für Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany.
Waldeck
W
W
D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002472:Cell Transformation, Viral; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry*; D008745:Methylation; D051379:Mice; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D002843:Chromatin; D004279:DNA, Viral; D009698:Nucleoproteins; D014764:Viral Proteins