Effects of DNA supercoiling on the topological properties of nucleosomes.

Abstract

In the nucleosome core particle, at least 145 base pairs of DNA are bound to the histone octamer in a superhelical conformation. We have asked what effect the presence of these particles has on the ability of DNA gyrase to supercoil DNA. Synthetic minichromosomes, constructed by reconstituting complexes of core histones with the closed circular plasmid pBR322, were treated with various amounts of DNA gyrase. We have found that the maximum level of supercoiling that is attainable is nearly identical for proteinfree plasmids and for plasmids half-saturated with core histones, even though supercoiling does not result in a loss of histones from the complex. It appears that, at sufficiently high levels of supercoiling, the core particle is disrupted in such a way that the DNA bound to histones is no longer constrained.Although there is considerable evidence linking the biological function of prokaryotic DNA to its topological state, the role of DNA supercoiling in eukaryotes is more obscure. Data from transfection experiments in eukaryotic cells suggest that expression ofgenes introduced on plasmids depends upon the presence of intact closed circular DNA (1). Other evidence suggests that chromatin domains, either genomic (2) or extrachromosomal (3), may exist in some circumstances in torsionally stressed states; it has been argued that such states are associated with expression ofgenes within the domain. In every case, however, the assessment of the role of supercoiling is complicated because of the presence of histones, which alter torsional stress when they bind to topologically isolated DNA domains.In the earliest experiments designed to measure the topological effects of histone binding, complexes of core histone octamers and relaxed closed circular DNA were incubated with topoisomerase I, and the resulting linking number change (AL) was measured after the DNA had been freed of protein (4). A recent determination by this method gives a value of AL = -1.0/core particle (5). The binding of a histone octamer thus has a topological effect equivalent to the unwinding of one turn of DNA duplex. Since the final linking number measurements were made after treatment with topoisomerase I, the observed values refer to a DNA-protein complex that is under no torsional stress.DNA within the nucleus is not necessarily in such a relaxed state, however. As noted above, there is some evidence that certain regions of the genome are under stress. This could in principle arise either through the removal ofhistone octamers from a topologically isolated domain or by the action of a putative DNA gyrase. In either case, the way in which the stress is distributed through the domain will be determined by the properties of the DNA in the remaining nucleosomes of the domain. Something is known of the effect of bound histone octamers on the torsional properties of the surrounding DNA when the DNA is relaxed (6), but there have been no measurements of the properties of DNA within nucleosomes associated with plasmids that are superhelic...

DNA linking potential generated by gyrase

“…Recent data on DNA minicircles showed the same maximum value of -oGyr [45]. In addition, it has been shown that oGYr does not change with increasing amount of gyrase added (starting from a specified value) [44]. This conclusion is supported by our experiments.…”

Bright Mutants ofVibrio fischeriES114 Reveal Conditions and Regulators That Control Bioluminescence and Expression of theluxOperon

“…(B) Supercoiling of plasmid DNA isolated from ES114 or topA mutants was assayed by electrophoresis through 0.8% agarose containing 20 g/ml chloroquine. DNA that is less negatively supercoiled prior to loading will migrate more rapidly at this concentration of chloroquine (29). Laddering on the gel is indicative of topoisomers of the same plasmid that differ by one linking number.…”

Structural plasticity of single chromatin fibers revealed by torsional manipulation

“…4b): ∆Lk ~ -1 obtained in higher salt against ∆Lk ~ -0.8 in B 0 (also favoring entry-exit DNA repulsion) [Interestingly, a similar ~+0.2 shift in ∆Lk was also measured in the minicircle system with acetylated mononucleosomes in phosphate, a buffer which further destabilizes histone tails/DNA interactions 9 .] ii) The property of reconstituted minichromosomes to withstand as much negative supercoiling (σ~-0.1) as the corresponding naked DNA upon treatment with DNA gyrase 35 , i.e. the nucleosome apparent "transparency" to that enzyme in spite of the trapping of most DNA in the nucleosome cores, must result from the shift of all nucleosomes to the negative state 9 , rather than from a forced undertwisting of the DNA on the histone surface 34 .…”