title: DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA.
abstract: The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ∼170 kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.
authors: Laval University Cancer Research Centre, 9 rue MacMahon, Québec QC G1R2J6, Canada.
authors: Sławomir
authors: S
authors: Kumala
authors: Yasmina
authors: Y
authors: Hadj-Sahraoui
authors: Joanna
authors: J
authors: Rzeszowska-Wolny
authors: Ronald
authors: R
authors: Hancock
mesh_terms: D002460:Cell Line; D002843:Chromatin / Q000737:chemistry*; D002875:Chromosomes / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D053837:DNA Cleavage; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis; D006801:Humans; D007202:Indicators and Reagents
chemical_list: D002843:Chromatin; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D004247:DNA; D004262:DNA Restriction Enzymes