Chromatin remodeling: insights and intrigue from single-molecule studies - PubMed (original) (raw)

Review

Chromatin remodeling: insights and intrigue from single-molecule studies

Bradley R Cairns. Nat Struct Mol Biol. 2007 Nov.

Abstract

Chromatin remodelers are ATP-hydrolyzing machines specialized to restructure, mobilize or eject nucleosomes, allowing regulated exposure of DNA in chromatin. Recently, remodelers have been analyzed using single-molecule techniques in real time, revealing them to be complex DNA-pumping machines. The results both support and challenge aspects of current models of remodeling, supporting the idea that the remodeler translocates or pumps DNA loops into and around the nucleosome, while also challenging earlier concepts about loop formation, the character of the loop and how it propagates. Several complex behaviors were observed, such as reverse translocation and long translocation bursts of the remodeler, without appreciable DNA twist. This review presents and discusses revised models for nucleosome sliding and ejection that integrate this new information with the earlier biochemical studies.

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Figures

Figure 1

Figure 1

Modes of nucleosome remodeling. Remodelers enable access to nucleosomal DNA through sliding, ejection or H2A-H2B dimer removal. The SWR1 complex is unique in its ability to efficiently replace H2A-H2B dimers with dimers bearing a histone H2A variant (Htz1 or H2A.Z). Act denotes an activator with a binding site on the nucleosome.

Figure 2

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 beads. 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,, only one of which is depicted. 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. 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 strands. See text for details. ss, single-stranded; ds, double-stranded; Dig, digoxygenin; Bio, biotin.

Figure 3

Figure 3

Models for DNA movement around nucleosomes. (a) Model for remodeling by ISWI, with contributions from many studies,,,,-. See text for details. A DNA loop is formed on the nucleosome surface by the concerted action of a DNA translocase (Tr) domain located near the dyad and a DBD located in the linker, near the nucleosome entry/exit site. DNA release by the Tr domain enables loop passage, and the complex then resets in the original position. (b) Structure of Rad54 ATPase domain with the two RecA-like regions in red and green. Interaction is limited to one face of the DNA, enabling interaction with nucleosomal DNA. (c) The 1+10 ratchet model, depicting the relative orientations of key domains on the nucleosome. The Tr domain remains in a fixed position relative to the histone octamer, whereas the DBD alternates between one of two conformations, through a flexible hinge (H). (d) The 1+10 ratchet model (see text). Triangle and square mark fixed positions as a reference for DNA movement relative to the stationary tracking domain and mobile DBD. c and d illustrate the same process: c shows top view and nucleosome context; d shows the movement of the DNA in the translocation cycle.

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