Figure 2

Single-molecule methods for observing remodeler translocation. (a) Magnetic tweezers tether a single DNA molecule between a glass slide and a magnetic bead. Magnets placed above the slide provide a calibrated stretching force (F_mag), and they can be rotated to cause bead rotation and the formation of plectonemes outside of the region bound by RSC (the formation of external plectonemes is not shown, but is discussed in the text). Translocation by RSC forms a constrained loop (which may include a plectoneme) and reduces the apparent end-to-end length of the DNA, as measured by the position of the bead (detected by video microscopy). See text for additional details. (b) Optical tweezers tether a single molecule of DNA (torsionally unconstrained) between two polystyrene beads⁵⁰. One bead is connected to a pipet, which is calibrated for movement along the axis parallel to the stretching force. The other bead is held by a laser (an optical trap), with the force needed to keep the bead in the center of the trap calibrated by laser power. After DNA has been stretched to a defined tension, remodeler action generates a DNA loop constrained to the nucleosome surface (Nuc), which shortens the distance between the beads, and the change in the pipet's position is directly measured. (c) Direct visualization of translocation. Two methods have been used^46,51, only one of which is depicted⁴⁶. In this method, a single DNA molecule is tethered at one end to a polystyrene bead by a biotin/streptavidin linkage, anchored by an optical trap and stretched by laminar flow. Yeast Rad54 was labeled with fluorescein isothiocyanate (FITC, via an antibody) and movement (+ATP) was visualized with an epifluorescence microscope. See ref. 51 for a description of a related and highly elegant technique involving stretched ‘DNA curtains’. (d) Analysis of nucleosome remodeling products by DNA unzipping. This technique reports the position of a nucleosome on a DNA fragment, and its structure, by measuring the force required to separate the two DNA strands⁵². See text for details. ss, single-stranded; ds, double-stranded; Dig, digoxygenin; Bio, biotin.