Real-Time Detection of Single-Molecule DNA Compaction by Condensin I Terence R Strick, Tatsuhiko Kawaguchi, Tatsuya Hirano  Current Biology  Volume 14, Issue 10, Pages 874-880 (May 2004) DOI: 10.1016/j.cub.2004.04.038

Figure 1 Experimental Setup and Topological Calibration (A) Experimental setup. A single, unnicked, dsDNA molecule is shown attached at one end to a glass surface and at the other to a 1 μm superparamagnetic bead. The {x, y, z} position of the bead above the surface was sampled at 30 Hz by using videomicroscopy, and can be determined to within about 10 nm with a ∼1 Hz bandwidth [19, 20]. This measurement yields the end-to-end extension, l, of the DNA. A pair of NdFeBr magnets located above the sample allows us to control the applied stretching force F and DNA supercoiling σ (see text for details). Addition of ATP and condensin to the system leads to physical DNA compaction; i.e., a reduction in DNA end-to-end extension. Two possible modes of condensin action are shown, including compaction by a single molecule or compaction driven by interactions between two DNA bound condensins (see Discussion for details). (B) Calibration of DNA topology. 11 kb DNA extension versus supercoiling behavior is measured in the enzyme's reaction buffer while holding the DNA at a constant force (F = 0.4 pN). Current Biology 2004 14, 874-880DOI: (10.1016/j.cub.2004.04.038)

Figure 2 Compaction of Torsionally Relaxed 11 kb DNA by Mitotic Condensin in the Presence of ATP The force is constant at F = 0.4 pN. Time traces showing the DNA extension as a function of time are presented with raw data in green and averaged data (∼1 s window) in red. (A) DNA extension versus time measured in the presence of mitotic condensin plus ATP (1 mM). (B) DNA extension versus time measured in the presence of mitotic condensin but no ATP. (C) DNA extension versus time measured in the presence of mitotic condensin plus AMP-PNP (1 mM). Current Biology 2004 14, 874-880DOI: (10.1016/j.cub.2004.04.038)

Figure 3 Detection and Analysis of Discrete Compaction/Decompaction Events by Condensin (A) Discrete and reversible compaction events can be observed on torsionally relaxed, stretched (F = 0.4 pN) 4 kb DNA in the presence of 0.2 mM ATP and equimolar amounts of condensin and competitor DNA in solution. Raw data points are in green and averaged data (∼1 s window) in red. (B) Histogram of compaction/decompaction step sizes measured from time traces obtained as in (A) (n = 228 points, mean = 80 nm, SD = 40 nm). (C) Histogram of compaction/decompaction step sizes measured from time traces obtained as in (A) but with negatively supercoiled DNA (σ = −0.018) (n = 131 points, mean = 75 nm, SD = 50 nm). (D) Histogram of compaction/decompaction step sizes measured from time traces obtained as in (A) but with positively supercoiled DNA (σ = 0.018) (n = 99 points, mean = 75 nm, SD = 45 nm). Current Biology 2004 14, 874-880DOI: (10.1016/j.cub.2004.04.038)

Figure 4 Dissociation of Condensin-DNA Complexes by a 10 pN Stretching Force (A) Condensin-DNA complexes are allowed to form on a torsionally relaxed, 11 kb DNA (σ = 0) at a force F = 0.4 pN in the presence of 1 mM ATP. At t = 0 the force is abruptly increased to 10 pN, driving dissociation of condensin-DNA complexes and a step-wise increase in DNA extension. Arrows indicate dissociation events. Raw data points are in green and averaged data (∼1 s window) in red. (B) Histogram of observed dissociation step sizes (n = 185 points, mean = 85 nm, SD = 110 nm). Current Biology 2004 14, 874-880DOI: (10.1016/j.cub.2004.04.038)