Condensin, a key component of the structure maintenance of chromosome (SMC) protein complexes, has recently been shown to be a motor that extrudes loops of DNA1. It remains unclear, however, how condensin complexes work together to collectively package DNA into chromosomes. Here we use time-lapse single-molecule visualization to study mutual interactions between two DNA-loop-extruding yeast condensins. We find that these motor proteins, which, individually, extrude DNA in one direction only are able to dynamically change each other’s DNA loop sizes, even when far apart. When they are in close proximity, condensin complexes are able to traverse each other and form a loop structure, which we term a Z-loop—three double-stranded DNA helices aligned in parallel with one condensin at each edge. Z-loops can fill gaps left by single loops and can form symmetric dimer motors that pull in DNA from both sides. These findings indicate that condensin may achieve chromosomal compaction using a variety of looping structures.
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Original imaging data and protein expression constructs are available upon request.
The Matlab source code used for quantification of the number of condensins and the DNA sizes is available at https://github.com/jacobkers/BN_CD18_EK_CondensinTrack.
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We thank S. Bravo, J. van der Torre and E. van der Sluis for technical support, and J. Eeftens, M. Ganji, A. Katan, B. Pradhan, B. Rowland, J.-K. Ryu and J. van der Torre for discussions. This work was supported by the Marie Skłodowska-Curie individual fellowship (to E.K.), the ERC grants SynDiv 669598 (to C.D.) and CondStruct 681365 (to C.H.H.), the Netherlands Organization for Scientific Research (NWO/OCW) as part of the Frontiers of Nanoscience and Basyc programs, and the European Molecular Biology Laboratory (EMBL).
The authors declare no competing interests.
Peer review information Nature thanks Leonid A Mirny, Jens Schmidt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, Geometry (top) and measurement scheme (bottom) of two-loop configuration. For quantification at each time point, we use the intensity profiles as schematized in the bottom panel. Loop content is counted from the excess peak intensity, whereas the tether section content is counted from the integrated intensity between background and tether level (red dashed lines). Tether sections labelled ‘left’, ‘gap’ and ‘right’ are bound by the intensity profile edges and loop positions (white dashed lines). All content is then converted to genomic content by scaling to the total counts above background (total coloured area). b, examples of quantification of the DNA gap between two loops when two condensins are oriented with their anchors facing towards each other, showing that the DNA lengths in between two loops stay constant. Data represent 11 independent experiments. c, d, Additional examples of kymographs and the corresponding DNA-loop-size estimations for two single loops. The data show examples of molecules that redistributed loop sizes (c) and an example of a molecule for which the loop size stayed unchanged (d). Representative of 14 (c) and 11 (d) independent experiments. e, More examples of simultaneous change of DNA-loop size for loop 2 versus for loop 1 (n = 16 molecules, 11 independent experiments). f, Amount of DNA-loop shrinkage as a function of DNA extension. g, Rate of DNA-loop shrinkage versus the distance between the two loops. h, Ratio of DNA molecules exhibiting loop shrinkage for different orientations of condensin anchors. Data are mean ± 95% confidence interval. n = 48 molecules from 14 independent experiments.
Extended Data Fig. 2 Quantification of DNA-loop slippage induced by an increase in DNA tension by application of a buffer flow and effect of ionic strength on loop slippage.
a, Snapshots of the DNA molecule from Supplementary Video 2 showing DNA slippage. Initially, a DNA loop is formed (25 s), which expands by reeling in DNA from the part of the tether above the loop (about 73 s), indicating that the anchor side of the condensin is clamped to the bottom part of the DNA. After stalling of the loop extrusion (91 s), a stronger buffer flow is suddenly applied (100 s), and consequently the loop was observed to shrink over time, indicating slippage of DNA through the condensin. Importantly, in this slippage process, no DNA is transferred from the loop to the DNA tether at the anchor side of the condensin, but all DNA is relaxed to the opposite (top) side of the DNA tether. b, Corresponding quantification of the distances between the stem of the DNA loop and the two tether ends, as indicated by the bars in in the top right image in a. While the distance between the bottom tether end and the loop stem (orange) stays approximately constant, the distance between loop stem and the top tether end (blue) clearly increases, indicating that loop slippage does not occur through the condensin anchor site but through its opposite side. c, Similar quantification of loop slippage for a different DNA molecule. Data in a–c represent three independent experiments. d, e, Kymograph (d) and extracted amount of DNA (e) within and between two loops, showing the diverging of the loops as these two condensins were oriented with their anchors facing each other, here occurring with loop slippage owing to reduced condensin anchoring strength in high-salt buffer (125 mM NaCl, 5 mM MgCl2). f, g, Rate (f) and amount (g) of DNA-loop shrinkage observed in low- (50 mM NaCl, 2.5 MgCl2) and high- (125 mM NaCl, 5 mM MgCl2) salt buffers. Box span 25th to 75th percentiles, centre line represents the median, and whiskers show maximum and minimum data points. P values are determined using two-tailed Student’s t-test.
a, b, Examples of kymographs of SxO-stained DNA and ATTO647N–condensin. The molecules in a show a gradual extension of a single loop into a Z-loop, and the simultaneous divergence of the locations of the two condensins that were previously co-localized near the single-loop region, into the edges of the Z-loop. b, Condensins are localized and fluctuating together with the edges of the Z-loop. Yellow arrows in a, b indicate the single-step photobleaching of the ATTO647N signals while the Z-loop remained stable. c, Examples of time traces of fluorescence intensities of individual ATTO647N-labelled condensin complexes near the edges of Z-loops that bleached in a single step-wise manner. An additional intensity is occasionally observed for short periods, owing to the temporary binding of an additional condensin. d, Single-step binding and the consequent bleaching event of the same condensin on a DNA molecule for which the initial single loop was extruded by an unlabelled condensin, enabling monitoring of the second condensin only. e, Two-step bleaching events observed for a case in which both condensins bleached while the Z-loop of DNA remained. f, Two-step binding event observed upon initiation of Z-loop formation. Blue arrows denote the bleaching events; orange arrows denote the arrival of the condensins. Data in a–f represent six independent experiments. g, Fluorescence intensity distributions for condensins that are temporarily bound on DNA but did not lead to loop extrusion (top), and for those that are located at Z-loop edges (bottom). h, Number of condensin complexes at each edge, estimated from the two histograms in g.
Extended Data Fig. 4 Additional data showing that Z-loops are made up of three parallel dsDNA molecules both under flow and in the absence of flow.
a, Snapshots (top) and the corresponding schematics (bottom) of Z-loops that were observed on different DNA molecules. By application of flow (direction indicated by the white arrows), we visualized three parallel dsDNA molecules that are linearly stretched along the DNA molecule. Representative of ten independent experiments. b, Snapshots and kymographs showing the formation of a single loop and the subsequent extension into a Z-loop in the absence of flow. Representative of 12 independent experiments. c, Time evolution (from orange to navy) of fluorescence intensity profiles extracted from five different lines of the kymograph in a. Each plot is normalized to the average DNA intensity in the non-looped region. During Z-loop formation, fluorescence intensity profiles initially display a sharp peak, characteristic of a single loop, which progressively broadens, until it levels off at about three times the intensity of the dsDNA outside the loop—consistent with three parallel dsDNA molecules. d, Normalized fluorescence intensity distributions extracted from fully stretched Z-loops for the regions inside (orange) and outside (blue) the Z-loops, showing an average value at onefold and threefold the single-DNA value, consistent with Z-loops consisting of three dsDNA stretches in parallel.
a, Probability of observing DNA molecules exhibiting a single loop, two separate loops and a Z-loop, measured at higher ionic concentration (125 mM NaCl, 5 mM MgCl2). The condensin concentration used was 10 nM. Data are mean ± s.d. from three independently measured datasets (ntot = 106 molecules). b, Plot of the lifetime of extruded Z-loops (defined as the time between the start of Z-loop extension and disruption into a single loop). About 50% of the extruded DNA loops did not release within the observation window of 200 s. ntot = 36 molecules from 12 independent experiments. c, Frequency of disruption events per DNA molecule that had two separate loops or a Z-loop, showing that Z-loops are more stable than two individual loops. Data are mean ± s.d. from 3 independent datasets (ntot = 99 molecules from 2 independent experiments). P values were determined by two-sided t-test.
Extended Data Fig. 6 Additional examples of DNA visualizations during Z-loop formation by extruding a loop within a loop or by merging of two parallel loops.
a–c, Additional examples of series of snapshots (top) and the corresponding schematics (bottom), showing transitions from single-loop extrusion to nested-loop formation to Z-loop extension. Initially, a single loop is extruded and enlarged until it approaches the tethered end of DNA. Subsequently, the single loop becomes further compacted, turning into the nested loop, followed by a gradual elongation into a Z-loop. In the events of Z-loop disruption (b, c), the structure reverts to a single loop, which is located either at the position where the initial single loop formed (b) (see also Fig. 2b) or at the newly formed edge of the Z-loop (c). The latter indicates that disruption occurred by release of the first condensin, after the second condensin had extended to the leading edge of the Z-loop structure. Note that the fact that Z-loops collapse, within a single frame, into a single loop also supports Z-loops comprising a single loop and a DNA segment underneath the loop. Data in a–c represent 20 independent experiments. d, Snapshots and schematics of two parallel loops that are individually formed (at 10 s) run into each other (about 99 s) and subsequently transform into a Z-loop (about 137 s), which is visualized by application of buffer flow (about 173 s). Representative of eight independent experiments.
Extended Data Fig. 7 Additional examples of visualization of DNA and condensins during Z-loop formation.
Snapshots of SxO-stained DNA (top), ATTO647N-labelled condensin (middle) and overlays (bottom), showing the locations of two condensins and the simultaneous transitions of DNA conformations during Z-loop formation. A second condensin binds at the location in the loop that is created by the first condensin, and approaches the first condensin while compacting the single loop further, whereupon one of the two condensins moves away from the other as the Z-loop extends. Schematics below the images provide visual guidance. Arrows denote the location of the two condensins. Representative of three independent experiments.
Extended Data Fig. 8 Further examples of visualization of DNA and condensins during Z-loop formation, demonstrating condensin traversals.
Snapshots of SxO-stained DNA (top), ATTO647N-labelled condensin (middle) and overlays (bottom), tracing the locations of the second condensin during Z-loop formation. In these examples, the fluorescence signal from the first condensin, which extruded the single DNA loop, was photobleached before the binding of the second condensin. Schematics below the images provide visual guidance. Representative of ten independent experiments.
a, Snapshots and fluorescence-intensity kymographs along the axis connecting both ends of the DNA tether (x axis) showing a transition of DNA conformation from bare DNA to a single loop to nested loops to a gradual extension of a Z-loop, and the related time lags. b, A further example of snapshots (left) and kymographs (right) for the DNA molecule shown in Fig. 2d. When the buffer flow was nearly perpendicular to the DNA tether, fluorescence intensity kymographs were obtained both along the axis parallel to the extruded single loop (X) and along the axis connecting both ends of the DNA tether (Y). Kymographs such as the ones in a, b were used to estimate the time lags and the DNA-loop-extrusion rate of loops within loops plotted in Fig. 2g, h. Data in a, b represent 20 independent experiments. c, Additional snapshots (left) and kymographs (right) for a DNA molecule that showed Z-loop formation at the third attempt to form a loop within a loop. Representative of three independent experiments. Among the molecules that formed nested loops (ntot = 34), 82% directly formed a Z-loop, 12% led to a Z-loop after 2–3 attempts to form a nested loop (such as in this example) and 6% of the cases did not lead to Z-loop formation. d, Comparison of lag time Δt2 for Z-loops pulling in DNA from one or two sides, showing no significant difference between Z-loops that gather DNA from one side and those that gather DNA from both sides (see Fig. 2g). Boxes span 25th to 75th percentiles, centre lines represent the median, and whiskers show maximum and minimum data points. P value was determined by two-sided t-test. e, f, Snapshots (left) and kymograph (right) showing the formation of a single loop and the subsequent extension into a Z-loop in the absence of buffer flow (e) and the amounts of DNA extracted from the kymograph for the loop region (II) and the regions outside the loop (I and III) (f). Dashed lines indicate the termination of the single loop growth (green) and the initiation of the Z-loop formation (orange). The rates of single loop extrusion and Z-loop growth are as shown in Fig. 2h; they were extracted by a simple linear fit to the linear rise in the first 10 s of data in traces like the one shown here. Data in e, f represent 12 independent experiments. g, Rate of loop growth versus DNA extension for single and Z-loops.
Extended Data Fig. 10 Effect of DNA compaction by Z-loops versus single loops measured for single-tethered and for double-tethered DNA molecules.
a, b, Example snapshots (left) and kymographs (right) for the single-tethered DNA molecules showing single loop (a) and Z-loops into which DNA is pulled from two sides (b). As in the kymograph shown in a, single loops grow either towards the direction of the free end or towards the tethered end, whereas the length of DNA strand on the opposite side remains constant. When, on reeling in DNA, the single loop reaches the free end of the DNA, it collapses within a single frame (white arrow). In the case of Z-loops into which DNA is pulled from both sides as shown in b, both sides of the DNA are shortened. When the Z-loop reaches the free end of the DNA, it collapses into a single loop within a single frame, near one of the edge of the Z-loop. These data for a single loop and a Z-loop reeling in DNA from two sides were measured at 1 nM and 10 nM condensin, respectively. Data represent two (a) and three (b) independent experiments, respectively. c, Visual explanation of how the final length of a Z-loop relates to the initial single loop size l in our double-tether DNA assay, where the condensins stall owing to high DNA tension. d, Final Z-loop size versus single-loop size before the Z-loop formation. A linear relation is observed, with 30% of molecules exhibiting the maximum possible Z-loop size, 1.5 times the size of the initial single loop (n = 32 molecules, 10 independent experiments). This size estimate of 1.5 times is determined by the nature of our specific set-up, in which loops reach only a finite size because of stalling, and therefore cannot be generalized. In our double-tether DNA assay, strain on the DNA increases rapidly as soon as the initial loop expands, making the first condensin typically inactive before the start of the Z-loop, thus limiting the length of the Z-loop to at most 1.5 times that of the initial loop. However, Z-loops can, in principle, grow infinitely long if no tension is built up during the growth of the loop, as shown in b. e, Visual explanation of the definition of maximum fold compaction (FC). f, g, Maximum fold compaction (f) and the corresponding stall force (g; estimated from the known force–extension relation28) for single loops (n = 49 molecules) and Z-loops (n = 22 molecules). The similar fold compaction, as measured in the double-tether assay, which exhibits a high internal tension, shows that two condensins forming a Z-loop do not linearly compact DNA much more than two condensins that form separate single loops in this condition. Data were obtained from ten independent datasets. Boxes span 25 to 75th percentiles, centre line represents the median and whiskers show maximum and minimum data points. h, Snapshots (left) and kymograph (middle) of a DNA molecule showing a Z-loop that exhibits random diffusion along the DNA tether, presumably owing to a ‘tug of war’ between the two partly slipping condensin motors, which occurs when Z-loops reeling in DNA from both sides fully extend along the double-tethered DNA. Representative of three independent datasets. Right, time traces of centre-of-mass positions of Z-loops that diffuse along DNA (n = 6 molecules from 3 independent datasets).
Supplementary discussion on comparison of the in vivo and in vitro condensin concentrations per DNA length.
Video showing mutual interactions between two DNA loop that are located far apart. A newly forming DNA loop that was extruded by the second condensin grew in intensity (i.e. loop size) at the expense of the original one, which concomitantly shrank in size over time. Representative of 16 independent experiments.
Video showing a single DNA loop that exhibits slippage when an increase in DNA tension is applied with a larger buffer flow. Notably, the slippage does not occur from the side with the anchor of condensin but mostly from the opposite side. Representative of 3 independent experiments.
Video showing a Z-loop structure that consists of three dsDNA stretches that are connected in parallel. Representative of 10 independent experiments.
Video showing the transition of DNA conformations from a bare DNA, to a single extruded loop, to a nested loop, to a Z-loop. Note that in between of 186s and 398s (where frames were cut for clarity – to avoid excessive waiting for the viewer) DNA stayed in its conformation of a fully extruded single loop. Representative of 20 independent experiments.
Video with overlaid channels of fluorescence from SxO-stained DNA (green) and ATTO647N-labeled condensin (red), showing a single loop that is extruded by a first condensin, binding of a second condensin within the loop, merging of two condensins accompanied by the formation of nested loop, followed by a diverging of the two condensins along the DNA as the Z-loop expands. Representative of 3 independent experiments.
Video with overlaid channels of fluorescence from SxO-stained DNA (green) and ATTO647N-labeled condensin (red), showing a single loop that is extruded by a first condensin, that was bleached before the binding of a second condensin at the DNA-loop region, whereupon the second condensin approached the DNA-loop stem, followed by a linear translocation along the DNA outside of the initial loop. Representative of 10 independent experiments.
Video showing a Z-loop that is formed by the merger of two single loops that encounter each other along the DNA. Representative of 8 independent experiments.
Video showing two-side-pulling Z-loop where DNA is reeled in symmetrically from both sides, leading to a simultaneous expansion of the Z-loop to both directions. Representative of 5 independent experiments.
Video showing two-side-pulling Z-loop where DNA is reeled in symmetrically from both sides, leading to a simultaneous expansion of the Z-loop to both directions. Representative of 5 independent experiments.
Video showing a Z-loop that fills the unextruded DNA gap between two single loops. Representative of 2 independent experiments.
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Kim, E., Kerssemakers, J., Shaltiel, I.A. et al. DNA-loop extruding condensin complexes can traverse one another. Nature 579, 438–442 (2020). https://doi.org/10.1038/s41586-020-2067-5
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