Real-time imaging of DNA loop extrusion by condensin

Abstract

It has been hypothesized that SMC protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We directly visualized the formation and processive extension of DNA loops by yeast condensin in real time. Our findings constitute unambiguous evidence for loop extrusion. We observed that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1500 base pairs per second, using the energy of adenosine triphosphate hydrolysis. Condensin-induced loop extrusion was strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization.

Fig. 1. Single-molecule assay for the visualization of condensin-mediated DNA looping.

(A) Cartoon representation of the S. cerevisiae condensin complex. (B) Side and top view schematics of DNA that is doubly tethered to a polyethylene glycol (PEG)-passivated quartz surface via streptavidin-biotin linkage. (C) Snap-shot of a double-tethered λ-DNA molecule (100 ms exposure) visualized by Sytox Orange (SxO) staining. Note the homogeneous fluorescence intensity distribution along the DNA. Dashed magenta circles indicate the surface attachment sites of the DNA. (D) Side and top view diagrams showing DNA loop formation on double-tethered DNA by condensin. (E) Snap-shot of condensin-mediated DNA loop formation at one spot (indicated by the yellow arrow) along a SxO-stained DNA molecule. (F) Strategy to visualize DNA loops. Application of flow perpendicular to the axis of the immobilized DNA extends the loop within the imaging plane. (G) Snap-shot of an extended DNA loop that is stretched out by flow (white arrow) perpendicular to the DNA, as illustrated in (F).

Fig. 2. Real-time imaging of DNA loop extrusion by condensin.

(A) Series of snap-shots showing DNA loop extrusion intermediates created by condensin on a SxO-stained double-tethered λ-DNA (Movie S3). A constant flow at a large angle to the DNA axis (white arrow) maintains the DNA in the imaging plane and stretches the extruded loop. A yellow arrow indicates the position of the loop base. At ~40 s, a small loop appears that grows over time until ~80 s, consistent with the loop extrusion model. A random linkage model would instead have predicted the sudden appearance of a loop that would have remained stable in size over time. After ~600 s, the loop suddenly disrupted. Schematic diagrams under each snap-shot are for visual guidance. (B) High time-resolution imaging reveals the splitting of the two DNA strands in the extruded loop in adjacent time frames.

Fig. 3. Loop extrusion is asymmetric and depends on ATP hydrolysis.

(A) Snap-shots showing the gradual extension of a DNA loop (yellow arrow) on a double-tethered λ-DNA molecule. (B) Kymograph of SxO fluorescence intensities shown in (A). (C) DNA lengths calculated from the integrated fluorescence intensities and the known 48.5-kbp length of the λ-DNA in the kymograph of panel (B) for regions outside the loop (I and III) and the loop region itself (II). (D–F) Fluorescence kymographs and intensity plots of a more stretched DNA molecule (end-to-end distance 9.1 μm) where the DNA loop stalls midway (D), of a DNA molecule where loop extrusion starts in the center and continues until reaching the physical barrier at attachment site (E), and of a loop-extrusion event that abruptly disrupts in a single step. (G) Kymograph and intensity plot for loop extrusion by a safety-belt condensin mutant complex, which displays dynamic changes of all three DNA regions and of the loop position. (H) Average loop extrusion rates (mean±SD) under various conditions. (I) Rate of loop extrusion plotted versus the relative DNA extension in relation to its 20-μm contour length. Filled circles are calculated from region II, open circles from region III, the line serves as a visual guide. (J) Rate of loop extrusion plotted versus the force exerted within the DNA due to increased DNA stretching upon increase of the loop size. The line serves as a visual guide.

Fig. 4. Loop extrusion is induced by a single condensin complex.

(A) Images of the same field of view of SxO-stained DNA (top left), ATTO647N-labeled condensin (top right) and their merge (bottom) reveal condensin at the stem of an extruded DNA loop (yellow arrow). Images are integrated over 2 s of a movie. (B) Kymographs of SxO-stained DNA (top left), ATTO647N- condensin (top right) and their merge (bottom left) of a real-time movie of DNA loop extrusion. The corresponding ATTO647N fluorescence time trace (bottom right) shows single-step binding and single-step photobleaching events of the DNA-bound condensin. (C) Fluorescence intensity distributions for condensin binding events that led to DNA-loop extrusion (left), condensin bleaching in such events (middle), and binding events that did not lead to loop extrusion (right) measured under similar optical conditions. (D) Histogram of the number of condensin complexes that show loop extrusion activity as counted from the fluorescence steps. (E) Model for DNA loop extrusion by condensin. One strand of DNA is anchored by the kleisin and HEAT-repeat subunits (yellow-orange) of the condensin complex, which extrudes a loop of DNA.