Nucleosome assembly depends on the torsion in the DNA molecule: a magnetic tweezers study

Abstract

We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers. To verify our hypothesis that the lack of full nucleosome assembly on topologically constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we carried out experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Indeed, such rotation did lead to the same nucleosome saturation level as in the case of nicked tethers. We conclude that levels of positive supercoiling in the range of 0.025-0.051 (most probably in the form of twist) stall the nucleosome assembly process.

Figure 1

Components used for assembly experiments. (a) Schematic for preparing the DNA construct: the three DNA fragments (dig-sticker, biotin-sticker, and 16.5 kb DNA from pxΔ2 DNA) were prepared separately (see Materials and Methods) and ligated together. (b) Purified histone octamer from chicken erythrocytes and purified recombinant yNap1 were analyzed by 15% SDS-PAGE gel. (c) Analysis of the nucleosome array reconstituted in bulk by 1% agarose gel.

Figure 2

(a) Schematic of the magnetic tweezers setup for studying nucleosome array assembly. A single double-stranded DNA molecule is suspended between a magnetic bead and the surface of the cuvette in a torsionally constrained manner. A mixture of core histones and histone chaperone Nap1 was injected into the cuvette to initiate assembly. The shortening in DNA extension due to formation of nucleosomes was measured by monitoring the movement of the bead in (z), as well as by Brownian motion analysis. (b) Rotation-extension curve on the torsionally constrained DNA at F = 0.3 pN. At low force, rotation of the two external magnets induced (+) or (−) supercoiling in the DNA molecule, which led to plectoneme formation, and thus shortening of the DNA tether. (c) Brownian motion scatter in (x,y) for the naked, relaxed DNA was larger than for the supercoiled DNA or reconstituted nucleosomal array.

Figure 3

Real-time assembly of nucleosomal arrays followed as decrease in DNA tether length (Δz) as a result of DNA wrapping around the histone octamer. The flow of histone octamers and Nap1 into the cuvette causes apparent shortening of the tether; once the flow was stopped, the bead went back to its original position. The further changes in Δz reflected array assembly. (a) Example assembly curve on torsionally constrained DNA. The Δz (2.37 μm) indicates partial (∼48%) assembly. Even after 50 min the assembly did not go to completion. (b) Example assembly curve on nicked DNA as control. Inset shows more complete assembly (Δz = 3.20 μm; 78% nucleosome saturation) in ∼4 min.

Figure 4

Assembly curves on torsionally constrained DNA after intermittent relief of the accumulating supercoiling stress by rotating the external magnetic field. (Top curve) The external magnets were rotated once after 20 min of assembly (52 clockwise rotations that neutralize the positive supercoiling accumulating in the DNA template). The mechanically induced relaxation led to further assembly (Δz = 3.5 μm). (Bottom curve) The magnets were rotated twice: at 20 min (52 negative turns) and at 40 min (20 negative turns). More complete assembly (Δz = 4.39 μm) was achieved in ∼93 min.