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.

Nucleosome-Positioning Sequence Repeats Impact Chromatin Silencing in Yeast Minichromosomes

“…Here we observed that the chromatin organization of the HML-E silencer previously mapped in the yeast genome (Weiss and Simpson 1998) is retained in the minichromosomes and is very similar to the structure and function of the silencer elements in the genome. Our high-resolution mapping of the yeast minichromosomes in the silent and active states with micrococcal nuclease clearly shows that the complete minichromosome was readily accessible to the nuclease in sharp contrast to viral minichromosomes in human cells (Kumala et al 2012). Moreover, in our experiments the silent chromatin appears to be digested notably faster by the MNase than the active chromatin ( Figure 3C) although the MNase cleavage patterns of the active chromatin and the silent chromatin are similar ( Figure 3, C and D).…”

Modeling the repair of DNA strand breaks caused by γ-radiation in a minichromosome

“…To study ionizing radiation-induced DNA damage and repair in vivo we performed experiments using the circular 172 kb long Epstein-Barr virus minichromosome which is stably maintained in ∼50 copies in cells of the Burkitt's lymphomaderived Raji cell line. This minichromosome has the same nucleosomal structure as human chromosomes and its different topological forms and fragments appearing after irradiation can be easily observed and quantitated by DNA pulsed field gel electrophoresis [5,6]. One of the characteristic features of this minichromosome, which we observed earlier [6], is that introduction of one DSB causes a change of chromatin structure that prevents induction of further DSBs in the same molecule.…”

Intercalation of small molecules into DNA in chromatin is primarily controlled by superhelical constraint

“…Therefore, our observations are particularly relevant in the context of the mechanism of transcriptional regulation by enhancers. Relaxation of topological constraints may not necessarily entail a positive effect on transcription in view of the loss DNase I and restriction endonuclease sensitivity following chromatin nicking [60,61], likely to disturb the regulation of transcriptional processes. Indeed, X-ray and gamma-ray irradiation of cells was shown to decrease, rather than increase, overall transcriptional activity [62,63].…”