MOLECULAR BIOLOGY

Real-time imaging of DNA loopextrusion by condensinMahipal Ganji,1 Indra A. Shaltiel,2* Shveta Bisht,2* Eugene Kim,1 Ana Kalichava,1

Christian H. Haering,2† Cees Dekker1†

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

The spatial organization of chromosomes isof paramount importance to cell biology.Members of the SMC (structural mainte-nance of chromosomes) family of proteincomplexes, including condensin, cohesin,

and the Smc5/6 complex, play vital roles in re-structuring genomes during the cellular life cycle(1–3). The principles by which SMC complexesachieve these fundamental tasks are still incom-pletely understood. Models based on randomcross-linking of DNA by pairwise interactions orconformational changes in theDNA superhelicityhave been proposed (4, 5). An alternative hypoth-esis suggested that SMC protein complexes pro-cessively enlarge small loops in the genome (6),which would elegantly explain how condensinmediates the formation of mitotic chromosomestructures observed in electronmicrographs anddeduced from Hi-C chromosome conformationmapping experiments (7, 8). Indeed, polymer sim-ulations showed that loop extrusion can, in prin-ciple, result in the efficient disentanglement andcompaction of chromatin fibers (9–11). The recentdiscovery that condensin exhibits DNA translocaseactivity (12) was consistent with, but did not pro-vide conclusive evidence for (13),DNA loopextrusion.Here, we visualized the formation of DNA loops

by the Saccharomyces cerevisiae condensin com-plex in real time (Fig. 1A). We tethered both endsof a double-stranded 48.5–kilobase pair (kbp)l-DNAmolecule to a passivated surface (14, 15),using the buffer flow rate to adjust the DNA end-to-end length to a distance much shorter thanits contour length (Fig. 1B).We then imagedDNAafter staining with Sytox Orange (SxO; Fig. 1Candmovie S1). Upon flushing in 1 nM condensin(12) and 5mMadenosine triphosphate (ATP), weobserved the accumulation of fluorescence den-sity at one spot along the length of the DNA (Fig. 1,

D and E, fig. S1, and movie S2). This finding showsthat condensin induces local compaction of DNA.To visualize the compacted DNA structures in

the imaging plane of themicroscope, we appliedflow at a large angle with respect to the double-tetheredDNA. This revealed that the bright spotsweremade up of extended pieces of DNA, consist-ent with single large DNA loops (Fig. 1, F and G,fig. S2, andmovie S3).We observednoDNA loopformation bywild-type condensin in the absenceof either ATP orMg2+, nor when we replaced ATPby thenonhydrolyzable analogsATPgSorAMPPNP,nor when we used a mutant condensin that isunable to bind ATP. Condensin hence createsDNA loops in a strictly ATP hydrolysis–dependentmanner, either by gradually extruding DNA orby randomly grabbing and linking two DNA loci.To distinguish between these two possibilities,

we monitored the looping process by real-timeimaging of the DNA while applying constantflow. This revealed the gradual appearance of aninitially weak increase in fluorescence intensityat a local spot that grew into an extended loopover time (Fig. 2A, fig. S3, andmovies S4 and S5),providing direct visual evidence of loop extrusionand ruling out the random cross-linking model.The extruded loopswere in general stable (fig. S4),although they occasionally disrupted spontane-ously in a single step (Fig. 2A andmovie S6). Sucha single-step disruption suggests that the DNAloop had been extruded by a single condensinunit that spontaneously let go of the loop, insteadof a multistep relaxation of the loop due to mul-tiple units. Imaging at higher temporal resolutionallowed us to resolve the two individual DNAstrands in the extruded loop in consecutive timeframes. This demonstrated that condensin hadextruded an actual loop rather than an inter-twined, supercoiled, or otherwise connectedstructure (Fig. 2B, fig. S5, and movie S7).To quantify the kinetics of loop extrusion, we

returned to imaging in the absence of flow. Weconstructed kymographs frommovieswhere theloop first nucleated as a single weak fluorescentspot that subsequently expanded in size over time(Fig. 3, A and B, and movie S8). We divided each

line of the kymograph into the regions outsidethe loop (I and III) and theDNA loop region itself(II) and calculated, for every frame, the DNAlengths from the fluorescence intensity in eachregion (Fig. 3C and fig. S6A). This revealed theextrusion of sizable amounts ofDNA into the loop(region II), ranging from 5 to 40 kbp (the upperlimit of our assay) before reaching a plateau (fig.S6, B and C). Simultaneously, the DNA contentof one of the two outside regions (III) decreasedby the same amount, whereas the DNA contentof the other outside region (I), surprisingly, didnot change at all during the loop extrusion pro-cess (Fig. 3, D to F). This result was consistent inall loop extrusion events that we analyzed quan-titatively (N = 36; fig. S6, B to D) and shows thatthe loop extrusion process is asymmetric—a find-ing that is in stark contrast to theoretical loop ex-trusion models that have so far been based ontwo linkedmotor domains that translocate alongthe DNA in opposite directions, thereby reelingin DNA symmetrically from both sides (9–11).The asymmetry can be explained if one site in

the condensin complex remains stably anchored totheDNAwhile itsmotor site translocates along thesameDNA. A candidate for such aDNAanchor siteis the charged groove formed by the Ycg1 HEAT-repeat and Brn1 kleisin subunits (16). When werepeated the loop extrusion experimentswith amu-tant condensin that is unable to firmly entrapDNAwithin the groove, the extruded loops no longerremained stable over time, but instead changedsize and frequently changed position (Fig. 3G andfig. S7), resulting in a DNA decrease in one outsideregion and an increase in the other outside re-gion (compare the green curves in Fig. 3, C to G).Furthermore, when we increased the NaCl andMg2+ concentrations of the buffer, we observed un-idirectional motion of the extruded loop (fig. S8).Theweakening of a stable anchoring point in bothexperiments is consistent with slippage of DNAthrough the safety belt. The latter finding alsoexplains the observation of condensin transloca-tion onDNAunder the samebuffer conditions (12).We can estimate the speed of DNA loop extru-

sion from the slopes of the initial linear part of theextrusion curves, which yield an average rate of0.6 kbp/s. As expected, this rate depends on theconcentration of ATP (fig. S9) and the ability ofcondensin to bind and hydrolyze ATP (Fig. 3H).Correlation with the end-to-end lengths of individ-ual DNA molecules showed that the speed andefficiency of loop extrusion strongly dependedon the relative extension of the DNA, with ratesof up to ~1.5 kbp/s and>80%of all DNAmoleculesdisplaying loop extrusion at lowerDNA extensions(Fig. 3I and fig. S10). Although a direct compar-ison with Hi-C experiments is impeded by thecomplexity of chromosomes in cells, it is none-theless remarkable that condensation rates cal-culated by this method for condensin II (0.1 to0.2 kbp/s) (17) or a bacterial SMCcomplex (0.9 kbp/s)(18) are of the same order of magnitude.We notethat our assay provides a direct measurementof the condensation rate by individual condensinmolecules, as it avoids any ambiguity aboutroadblocks ormultiple condensin units working

RESEARCH

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1Department of Bionanoscience, Kavli Institute of NanoscienceDelft, Delft University of Technology, Delft, Netherlands. 2CellBiology and Biophysics Unit, Structural and Computational BiologyUnit, European Molecular Biology Laboratory, Heidelberg, Germany.*These authors contributed equally to this work.†Corresponding author. Email: christian.haering@embl.de (C.H.H.);c.dekker@tudelft.nl (C.D.)

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in series or in parallel at different positions onthe DNA.The dependence of the extrusion rate on the

DNA end-to-end length can be understood as aforce dependence: As the loop is extruded, theamount of DNA between the two attachmentpoints (excluding the loop) decreases correspond-ingly, thus continuously increasing the tensionwithin the DNA (19) (to 1.2 ± 0.5 pN;N = 23; seesupplementary materials) until the loop extru-sion process stalls (see, for example, Fig. 3D).Weused the known force-extension relation for DNAto plot the loop extrusion rate versus force, whichrevealed a steep dependence on force (Fig. 3Jand fig. S11). This very strong force dependence,where the loop extrusion rate drops substantiallynear 0.4 pN, is consistent with previousmagnetictweezer experiments (20–22), which also reportedcondensation rates thatmatch the loop extrusionrates that wemeasured at similar forces (Fig. 3J).Together, these findings show that condensin is afast, yet weak, loop-extruding motor that rapidlystalls against a modest force applied to the DNA.We measured an ATP hydrolysis rate of ~2

molecules/s for condensin in the presence of DNA(fig. S12A). Notably, such a bulk estimate only pro-vides a lower limit of the adenosine triphosphatase(ATPase) activity of condensin complexes in theactive loop extrusion process, because non–DNA-bound condensin molecules and complexes that

have stalled on the DNA are included in theseassays. If we nonetheless assume, for the sakeof argument, that condensin hydrolyzes two ATPmolecules in the course of extruding ~110 nm(~0.6 kbp) of the folded DNA every second, itwould take discrete condensation steps on theorder of the 50-nm size of a single condensincomplex. Scenarios that explain such large stepsmust be very different from those for conven-tional DNA motor proteins that move in single–base pair increments (23). An accurate modelwould need to take into account the flexibilityof DNA, such that for low forces, condensin canreach nearby spots on the folded DNA to occasional-ly capture and extrude much longer stretches ofDNA than the condensin size itself (12, 24, 25).This would be consistent with the very strong forcedependence of the extrusion rate that we observe.Finally, we labeled purified condensin holocom-

plexeswith a singleATTO647N fluorophore (fig. S12)and co-imaged them with SxO-labeled DNA in aHILO (highly inclined and laminated optical) sheetmode (26). As expected, ATTO647N-condensinfrequently localized to the stem of the extrudedDNA loop (Fig. 4A, fig. S13, andmovies S9 andS10).Using real-time monitoring, we consistently ob-served DNA binding of condensin in a single-stepappearance, followed by DNA loop extrusion andfinally single-step bleaching (Fig. 4B and figs. S14and S15; N = 20). This sequence of events un-

ambiguously demonstrates that the DNA loop wasextrudedbya single condensincomplex.Kymographsalso revealed another class of condensin binding andunbinding events with a short dwell time that didnot lead to DNA loop extrusion (fig. S16A). Thesewere the only DNA binding events in the absenceofATP (fig. S16B). Furthermore,weobserved eventswhere aDNA loopdisruptedwhile condensin stayedbound to theDNAand later started to extrudeanewDNA loop (fig. S14, D and E). Hence, during the pro-cess of loop disruption, condensin spontaneouslyreleases the extruded loop rather than dissociatingfrom the DNA.Quantification of the ATTO647N-condensin

intensity provided further evidence that it is asingle condensin complex that locates to the stemof the DNA loop. We compared the fluorescenceintensity for three cases: (i) condensin bindingevents that led to DNA loop extrusion, (ii) con-densinbleachingevents, and (iii) temporarybindingevents that did not lead to loop extrusion. All threecases revealed the same change in intensity (Fig. 4C),showing that a single condensin complex localizesto the stem of the DNA loop (Fig. 4D). If two con-densin complexes had assembled at the loop stem,the expected result would be a bimodal intensitydistribution with two peaks of similar heightfor single- and double-labeled condensin dimers(based on a labeling efficiency of 60 to 85%; seesupplementary materials). Instead, we observed

Ganji et al., Science 360, 102–105 (2018) 6 April 2018 2 of 4

Fig. 1. Single-molecule assay for thevisualization of condensin-mediated DNAlooping. (A) Cartoon representation ofthe S. cerevisiae condensin complex.(B) Side- and top-view schematics of DNAthat is doubly tethered to a polyethyleneglycol (PEG)–passivated quartz surface viastreptavidin-biotin linkage. (C) Snapshotof a double-tethered l-DNA molecule(exposure, 100 ms) visualized by SytoxOrange (SxO) staining. Note the homogeneousfluorescence intensity distribution alongthe DNA. Dashed magenta circles indicatethe surface attachment sites of the DNA.(D) Side- and top-view diagrams showingDNA loop formation on double-tethered DNAby condensin. (E) Snapshot of condensin-mediated DNA loop formation at one spot(indicated by the yellow arrow) along aSxO-stained DNA molecule. (F) Strategy tovisualize DNA loops. Application of flowperpendicular to the axis of the immobilizedDNA extends the loop within the imagingplane. (G) Snapshot of an extended DNA loopthat is stretched out by flow (white arrow)perpendicular to the DNA, as illustrated in (F).

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Ganji et al., Science 360, 102–105 (2018) 6 April 2018 3 of 4

Fig. 3. Loop extrusion is asymmetric anddepends on ATP hydrolysis. (A) Snapshotsshowing the gradual extension of a DNA loop(yellow arrow) on a double-tethered l-DNAmolecule. (B) Kymograph of SxO fluorescenceintensities shown in (A). (C) DNA lengthscalculated from the integrated fluorescenceintensities and the known 48.5-kbp length ofthe l-DNA in the kymograph of (B) for regionsoutside the loop (I and III) and the loop regionitself (II). (D to F) Fluorescence kymographs(left) and intensity plots (right) of a morestretched DNA molecule (end-to-end distance9.1 mm) where the DNA loop stalls midway(D), of a DNA molecule where loop extrusionstarts in the center and continues until reachingthe physical barrier at the attachment site(E), and of a loop extrusion event that abruptlydisrupts in a single step. (G) Kymograph andintensity plot for loop extrusion by a safety-beltcondensin mutant complex, which displaysdynamic changes of all three DNA regions andof the loop position. (H) Average loop extrusionrates (mean ± SD) under various conditions;WT, wild type. (I) Rate of loop extrusion versusrelative DNA extension in relation to its 20-mmcontour length. Solid circles are calculatedfrom region II, open circles from region III; theline serves as a visual guide. (J) Rate of loopextrusion plotted versus the force exertedwithin the DNA as a result of increased DNAstretching upon increase of the loop size.The line serves as a visual guide.

Fig. 2. Real-time imaging of DNA loopextrusion by condensin. (A) Seriesof snapshots showing DNA loop extrusionintermediates created by condensin on aSxO-stained double-tethered l-DNA (movieS3). A constant flow at a large angle tothe DNA axis (white arrow) maintains theDNA in the imaging plane and stretchesthe extruded loop. A yellow arrow indicatesthe position of the loop base. At ~40 s, a smallloop appears that grows over time until~80 s, consistent with the loop extrusionmodel. A random linkage model would insteadhave predicted the sudden appearance of aloop that remains stable in size over time.After ~600 s, the loop suddenly disrupted.Schematic diagrams under each snapshot arefor visual guidance. (B) Imaging at hightime resolution reveals the splitting of thetwo DNA strands in the extruded loop inadjacent time frames.

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no two-step bleaching events, a majority fraction(33/40) of single-step bleaching events, and asmall fraction of DNA molecules (7/40) thatshowed DNA loop extrusion without any visiblefluorescence (fig. S17), as expected for a labelingefficiency of 82%.Although SMC complexes are vital for chromo-

some organization in all domains of life, the molec-ular basis for their action had remained largelyspeculative. Our experiments unambiguously dem-onstrate that condensin exhibitsDNA loop extrusion

activity. Our real-time single-molecule dual-colormovies of condensin andDNA reveal that loops areextruded by a single condensin complex at the stemof the loop in a manner that is ATP hydrolysis–dependent, strictly asymmetric, andhighly sensitiveto forces applied to theDNA.Theseproperties can beexplained by a model (Fig. 4E) where condensinmakes stable contact with DNA at a binding siteand thenreels inDNAfromonlyoneside.The visualproof that condensin is a loop-extruding enzymereveals the key principle that underlies the orga-

nization of genome architecture by condensinandmost likely all other SMC protein complexes.

REFERENCES AND NOTES

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2162–2168 (2016).12. T. Terakawa et al., Science 358, 672–676 (2017).13. K. Nasmyth, Science 358, 589–590 (2017).14. M. Ganji, S. H. Kim, J. van der Torre, E. Abbondanzieri,

C. Dekker, Nano Lett. 16, 4699–4707 (2016).15. S. H. Kim, M. Ganji, J. van der Torre, E. Abbondanzieri,

C. Dekker, DNA sequence encodes the position ofDNA supercoils. bioRxiv 2017/08/24/180414 (2017).

16. M. Kschonsak et al., Cell 171, 588–600.e24 (2017).17. J. H. Gibcus et al., Science 359, eaao6135 (2018).18. X. Wang, H. B. Brandão, T. B. K. Le, M. T. Laub, D. Z. Rudner,

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(2017).22. T. R. Strick, T. Kawaguchi, T. Hirano, Curr. Biol. 14, 874–880 (2004).23. W. Yang, Annu. Rev. Biophys. 39, 367–385 (2010).24. J. Lawrimore, B. Friedman, A. Doshi, K. Bloom, Cold Spring

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159–161 (2008).

ACKNOWLEDGMENTS

We thank M. Kschonsak for providing the safety-belt mutant condensincomplex, J. Kerssemakers and J. van der Torre for technical support,and J. Eeftens, A. Katan, J.-K. Ryu, and E. van der Sluis for discussions.Funding: Supported by ERC grants SynDiv 669598 and CondStruct681365, the NetherlandsOrganization for Scientific Research (NWO/OCW)as part of the Frontiers of Nanoscience program and a Rubicon Grant,and the European Molecular Biology Laboratory. Author contributions:M.G. and C.D. designed the single-molecule visualization assay, M.G., E.K.,and A.K. performed the imaging experiments, I.A.S. and S.B. developedcondensin fluorescence labeling strategies and purified protein complexes,I.A.S. performed ATPase assays, C.D. and C.H.H. supervised the work,M.G., C.H.H., and C.D. wrote the manuscript with input from all authors.Competing interests: All authors declare that they have no competinginterests. Data and materials availability: Original imaging data andprotein expression constructs are available upon request.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/360/6384/102/suppl/DC1Materials and MethodsFigs. S1 to S17Movies S1 to S10References (27, 28)

17 December 2017; accepted 6 February 2018Published online 22 February 201810.1126/science.aar7831

Ganji et al., Science 360, 102–105 (2018) 6 April 2018 4 of 4

Fig. 4. Loop extrusion is induced by a single condensin complex. (A) Images of the same fieldof 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 amovie. (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 timetrace (bottom right) shows single-step binding and single-step photobleaching events of the DNA-boundcondensin. (C) Fluorescence intensity distributions for condensin binding events that led to DNA-loopextrusion (left), condensin bleaching in such events (center), and binding events that did not lead to loopextrusion (right) measured under similar optical conditions. (D) Histogram of the number of condensincomplexes that show loop extrusion activity, as counted from the fluorescence steps. (E) Model for DNA loopextrusion 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.

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Real-time imaging of DNA loop extrusion by condensinMahipal Ganji, Indra A. Shaltiel, Shveta Bisht, Eugene Kim, Ana Kalichava, Christian H. Haering and Cees Dekker

originally published online February 22, 2018DOI: 10.1126/science.aar7831 (6384), 102-105.360Science 

, this issue p. 102Scienceextrusion mechanism for chromosome organization.asymmetrically, with condensin reeling in only one end of the DNA. These data provide unambiguous evidence of a loopcondensin-mediated, adenosine triphosphate-dependent, fast DNA loop extrusion process. Loop extrusion occurred

visualized in real time aet al.extrusion has not been observed directly. Using single-molecule imaging, Ganji hypothesized to extrude DNA loops. Condensin has been shown to exhibit a DNA-translocating motor function, but

To spatially organize chromosomes, ring-shaped protein complexes including condensin and cohesin have beenLive imaging of DNA loop extrusion

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