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Real-time imaging of DNA loop extrusion by condensin

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Science  06 Apr 2018:
Vol. 360, Issue 6384, pp. 102-105
DOI: 10.1126/science.aar7831
  • 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) Snapshot of a double-tethered λ-DNA molecule (exposure, 100 ms) 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) Snapshot 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) Snapshot 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 snapshots 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 remains stable in size over time. After ~600 s, the loop suddenly disrupted. Schematic diagrams under each snapshot are for visual guidance. (B) Imaging at high time resolution 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) Snapshots 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 (B) for regions outside the loop (I and III) and the loop region itself (II). (D to F) Fluorescence kymographs (left) and intensity plots (right) 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 the 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; WT, wild type. (I) Rate of loop extrusion versus relative DNA extension in relation to its 20-μm contour length. Solid 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 as a result of 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 (center), 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.

Supplementary Materials

  • Real-time imaging of DNA loop extrusion by condensin

    Mahipal Ganji, Indra A. Shaltiel, Shveta Bisht, Eugene Kim, Ana Kalichava, Christian H. Haering, Cees Dekker

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S17
    • Captions for movies S1 to S10
    • References

    Images, Video, and Other Media

    Movie S1
    Movie showing homogenously distributed SxO fluorescence intensity along the length of a double-tethered λ-DNA molecule in the absence of condensin.
    Movie S2
    Movie showing DNA compaction by condensin. Compaction can be identified as a bright fluorescence spot within the SxO-stained DNA.
    Movie S3
    Movie showing DNA that has been compacted by condensin. Compaction can be identified as a bright fluorescence spot within the SxO-stained DNA. During this movie, a perpendicular flow is applied to reveal that the bright spot constitutes an extended DNA loop.
    Movie S4
    Movies showing DNA loop extrusion on SxO-stained DNA under a constant flow. In both movies, DNA initially displays an inverted U shape due to the applied flow. As the movies proceed, a bright fluorescence spot appears that grows into an extended loop, which finally stalls. At its maximum size, the DNA molecule appears as an inverted Y shape.
    Movie S5
    Movies showing DNA loop extrusion on SxO-stained DNA under a constant flow. In both movies, DNA initially displays an inverted U shape due to the applied flow. As the movies proceed, a bright fluorescence spot appears that grows into an extended loop, which finally stalls. At its maximum size, the DNA molecule appears as an inverted Y shape.
    Movie S6
    Movie that shows single-step disruption of the DNA loops at the end of movie S5, resulting in the return of a homogenous DNA intensity that was identical to the bare DNA at the start of the experiment..
    Movie S7
    Movie showing condensin-mediated DNA compaction under constant flow perpendicular to DNA. Near the end of the movie, the DNA loop splits, most likely due to a photo-induced double strand break. This rare event shows the two DNA strands that form the extruded loop.
    Movie S8
    Movie of DNA loop extrusion on double-tethered DNA in the absence of flow. The DNA initially exhibits a homogenous fluorescence intensity along its length. As time progresses, a bright fluorescence spot appears that migrates towards one of the two ends of the attached DNA.
    Movie S9
    Movie with overlaid channels of fluorescence from SxO-stained DNA (green) and ATTO647N-labeled condensin (red), showing condensin localization to the stem of the extruded DNA loop.
    Movie S10
    Movie with overlaid channels of fluorescence from SxO-stained DNA (green) and ATTO647N-labeled condensin (red), showing the correlative spatial fluctuations of the ATTO647N condensin signal and the SxO DNA loop.

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