Skip to main content

Advertisement

SpringerLink
Log in

Condensin-driven loop extrusion on supercoiled DNA

  • Article
  • Published:

From Nature Structural & Molecular Biology

View current issue Submit your manuscript

Abstract

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, for example, the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping are stimulated by positively supercoiled DNA, and condensin preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1: Condensin-mediated loop extrusion collects and stabilizes plectonemes into a single supercoiled DNA loop.
Fig. 2: Condensin loads near a plectoneme tip and moves downward along the plectoneme upon loop extrusion.
Fig. 3: Condensin is located at the stem or at the middle of a plectoneme after extrusion of a supercoiled loop.
Fig. 4: Positive supercoiling promotes condensin loading and loop formation, whereas negative supercoiling does not.
Fig. 5: DNA supercoiling generation by condensin and ATP.
Fig. 6: Recruitment of additional condensins onto a supercoiled loop.
Fig. 7: Model for condensin-mediated loop extrusion on supercoiled DNA.

Similar content being viewed by others

Data availability

Source data files are available for Figs. 1e,g,h; 2c,e; 3g; 4a,b; 5b–d and 6g,h and Extended Data Figs. 1; 3b; 4a,b; 5c and 10a,b. Original imaging data are available upon request. Source data are provided with this paper.

Code availability

The Python-based data analysis source code used for the analysis of the imaging data is available at https://github.com/biswajitSM/LEADS.

References

  1. Yatskevich, S., Rhodes, J. & Nasmyth, K. Organization of chromosomal DNA by SMC complexes. Annu. Rev. Genet. 53, 445–482 (2019).

    Article  CAS  Google Scholar 

  2. Ganji, M. et al. Real-time imaging of DNA loop extrusion by condensin. Science 360, 102–105 (2018).

    Article  CAS  Google Scholar 

  3. Davidson, I. F. et al. DNA loop extrusion by human cohesin. Science 366, 1338–1345 (2019).

    Article  CAS  Google Scholar 

  4. Kim, Y., Shi, Z., Zhang, H., Finkelstein, I. J. & Yu, H. Human cohesin compacts DNA by loop extrusion. Science 366, 1345–1349 (2019).

    Article  CAS  Google Scholar 

  5. Golfier, S., Quail, T., Kimura, H. & Brugués, J. Cohesin and condensin extrude DNA loops in a cell-cycle dependent manner. eLife 9, e53885 (2020).

    Article  Google Scholar 

  6. Kim, E., Kerssemakers, J., Shaltiel, I. A., Haering, C. H. & Dekker, C. DNA-loop extruding condensin complexes can traverse one another. Nature 579, 438–442 (2020).

    Article  CAS  Google Scholar 

  7. Baxter, J. et al. Positive supercoiling of mitotic DNA drives decatenation by topoisomerase II in eukaryotes. Science 331, 1328–1332 (2011).

    Article  CAS  Google Scholar 

  8. Sutani, T. et al. Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation. Nat. Commun. 6, 7815 (2015).

    Article  Google Scholar 

  9. Minchell, N. E., Keszthelyi, A. & Baxter, J. Cohesin causes replicative DNA damage by trapping DNA topological stress. Mol. Cell 78, 739–751.e8 (2020).

    Article  CAS  Google Scholar 

  10. Canela, A. et al. Genome organization drives chromosome fragility. Cell 170, 507–521.e18 (2017).

    Article  CAS  Google Scholar 

  11. Kimura, K. & Hirano, T. ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation. Cell 90, 625–634 (1997).

    Article  CAS  Google Scholar 

  12. Kimura, K., Rybenkov, V. V., Crisona, N. J., Hirano, T. & Cozzarelli, N. R. 13S condensin actively reconfigures DNA by introducing global positive writhe. Cell 98, 239–248 (1999).

    Article  CAS  Google Scholar 

  13. Bazett-Jones, D. P., Kimura, K. & Hirano, T. Efficient supercoiling of DNA by a single condensin complex as revealed by electron spectroscopic imaging. Mol. Cell 9, 1183–1190 (2002).

    Article  CAS  Google Scholar 

  14. Kimura, K., Cuvier, O. & Hirano, T. Chromosome condensation by a human condensin complex in Xenopus egg extracts. J. Biol. Chem. 276, 5417–5420 (2001).

    Article  CAS  Google Scholar 

  15. Eeftens, J. M. et al. Real‐time detection of condensin‐driven DNA compaction reveals a multistep binding mechanism. EMBO J. 36, 3448–3457 (2017).

    Article  CAS  Google Scholar 

  16. Gutierrez-Escribano, P. et al. Purified Smc5/6 complex exhibits DNA substrate recognition and compaction. Mol. Cell 80, 1039–1054.e6 (2020).

    Article  CAS  Google Scholar 

  17. Sun, M., Nishino, T. & Marko, J. F. The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation. Nucleic Acids Res. 41, 6149–6160 (2013).

    Article  CAS  Google Scholar 

  18. Ganji, M., Kim, S. H., van der Torre, J., Abbondanzieri, E. & Dekker, C. Intercalation-based single-molecule fluorescence assay to study DNA supercoil dynamics. Nano Lett. 16, 4699–4707 (2016).

    Article  CAS  Google Scholar 

  19. Kim, S. H. et al. DNA sequence encodes the position of DNA supercoils. eLife 7, e36557 (2018).

    Article  Google Scholar 

  20. van Loenhout, M. T. J., de Grunt, M. V. & Dekker, C. Dynamics of DNA supercoils. Science 338, 94–97 (2012).

    Article  Google Scholar 

  21. Ganji, M. et al. Real-time imaging of DNA loop extrusion by condensin. Science 360, 102–105 (2018).

    Article  CAS  Google Scholar 

  22. Thundat, T. et al. Atomic force microscopy of DNA on mica and chemically modified mica. Scanning Microsc. 6, 2 (1992).

    Google Scholar 

  23. Balke, V. L. & Gralla, J. D. Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli. J. Bacteriol. 169, 4499–4506 (1987).

    Article  CAS  Google Scholar 

  24. Freeman, L., Aragon-Alcaide, L. & Strunnikov, A. The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J. Cell Biol. 149, 811–824 (2000).

    Article  CAS  Google Scholar 

  25. Brandão, H. B. et al. RNA polymerases as moving barriers to condensin loop extrusion. Proc. Natl Acad. Sci. 116, 20489–20499 (2019).

    Article  Google Scholar 

  26. Johzuka, K., Terasawa, M., Ogawa, H., Ogawa, T. & Horiuchi, T. Condensin loaded onto the replication fork barrier site in the rRNA gene repeats during S phase in a FOB1-dependent fashion to prevent contraction of a long repetitive array in Saccharomyces cerevisiae. Mol. Cell. Biol. 26, 2226–2236 (2006).

    Article  CAS  Google Scholar 

  27. Jeppsson, K. et al. The chromosomal association of the Smc5/6 complex depends on cohesion and predicts the level of sister chromatid entanglement. PLoS Genet. 10, e1004680 (2014).

    Article  Google Scholar 

  28. Guo, M. S., Kawamura, R., Littlehale, M. L., Marko, J. F. & Laub, M. T. High-resolution, genome-wide mapping of positive supercoiling in chromosomes. eLife 10, e67236 (2021).

    Article  CAS  Google Scholar 

  29. Marko, J. F. & Neukirch, S. Competition between curls and plectonemes near the buckling transition of stretched supercoiled DNA. Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys. 85, 011908 (2012).

    Article  Google Scholar 

  30. Racko, D., Benedetti, F., Dorier, J. & Stasiak, A. Transcription-induced supercoiling as the driving force of chromatin loop extrusion during formation of TADs in interphase chromosomes. Nucleic Acids Res. 46, 1648–1660 (2018).

    Article  CAS  Google Scholar 

  31. Naughton, C. et al. Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures. Nat. Struct. Mol. Biol. 20, 387–395 (2013).

    Article  CAS  Google Scholar 

  32. Achar, Y. J., Adhil, M., Choudhary, R., Gilbert, N. & Foiani, M. Negative supercoil at gene boundaries modulates gene topology. Nature 577, 701–705 (2020).

    Article  CAS  Google Scholar 

  33. Rao, S. S. P. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014).

    Article  CAS  Google Scholar 

  34. Benedetti, F., Dorier, J. & Stasiak, A. Effects of supercoiling on enhancer–promoter contacts. Nucleic Acids Res. 42, 10425–10432 (2014).

    Article  CAS  Google Scholar 

  35. Nolivos, S. et al. MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation. Nat. Commun. 7, 10466 (2016).

    Article  CAS  Google Scholar 

  36. Sen, N. et al. Physical proximity of sister chromatids promotes Top2-dependent intertwining. Mol. Cell 64, 134–147 (2016).

    Article  CAS  Google Scholar 

  37. Japaridze, A. et al. Hyperplectonemes: a higher order compact and dynamic DNA self-organization. Nano Lett. 17, 1938–1948 (2017).

    Article  CAS  Google Scholar 

  38. Krull, A., Buchholz, T.-O. & Jug, F. Noise2Void—learning denoising from single noisy images. In Proc. IEEE/CVF Conf. Comput. Vis. Pattern Recognit. 2124–2132 (IEEE, 2019).

  39. Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17, 261–272 (2020).

    Article  CAS  Google Scholar 

  40. Lyubchenko, Y. L. & Shlyakhtenko, L. S. AFM for analysis of structure and dynamics of DNA and protein–DNA complexes. Methods 47, 206–213 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank E. van der Sluis and A. van den Berg for protein purification, and J. Kerssemakers, J.-K. Ryu, A. Katan, R. Barth, M. Tisma and L. van Eendenburg for discussions. C.D. was supported by the European Research Council (ERC) Advanced Grant 883684 (DNA looping), Netherlands Organization for Scientific Research (NWO) grant OCENW.GROOT.2019.012, and the NanoFront and BaSyC programs.

Author information

Author notes

  1. Eugene Kim & Biswajit Pradhan

    Present address: Max Planck Institute of Biophysics, Frankfurt am Main, Germany

Authors and Affiliations

  1. Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, Netherlands

    Eugene Kim, Alejandro Martin Gonzalez, Biswajit Pradhan, Jaco van der Torre & Cees Dekker

Authors
  1. Eugene Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alejandro Martin Gonzalez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Biswajit Pradhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jaco van der Torre
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cees Dekker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.K. and C.D. designed the single-molecule imaging experiments, E.K. performed the imaging experiments and analyzed the imaging data, A.M.G. performed the AFM experiments and analyzed the AFM data, B.P. contributed in image analyses, J.v.d.T. prepared the DNA construct, C.D. supervised the work, and all authors wrote the manuscript.

Corresponding author

Correspondence to Cees Dekker.

Ethics declarations

Competing interests

All authors declare that they have no competing interests.

Peer review

Peer review information

Nature Structural and Molecular Biology thanks Alice Pyne, Anders Hansen, and Jan Lipfert for their contribution to the peer review of this work. Primary Handling Editor: Carolina Perdigoto, in collaboration with the Nature Structural and Molecular Biology team. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Validation of our supercoiling generation assay.

To confirm the handedness of supercoiling, we introduced Topoisomerase I from E. coli, which only relaxes negative supercoils, to our DNA molecules on the surface that were generated to have positive/negative supercoils via SxO intercalation reaction. For negative supercoils, all the DNA molecules relaxed within 5 minutes of incubation with Topo1, while in the case of positive supercoils, the amount of supercoiled molecules only decreased by 25. Since, independent of Topo1, DNA molecules can be also nicked by exposure of excitation laser (which would resolve the supercoiling), the actual fraction of positive supercoiled DNA in our assay should be well above 75%. These data confirm the handedness of the supercoiling generated via SxO intercalation.

Source data

Extended Data Fig. 2 Fluorescence-intensity kymographs showing additional examples of loop extrusion events on supercoiled DNA.

(a) Examples showing condensin collects all the plectonemes upon loop extrusion and stabilizes them at its location. Yellow arrows show the location of the start of the gradually growing peak during loop extrusion. (b) Examples showing cases where condensin loop extrusion did not lead to absorption of all the plectonemes. In these events (11% of the total cases, Ntot=36), condensin was bound relatively close to the tethered end of DNA and the DNA loop extrusion was limited by reaching the end position of the DNA. Yellow arrows show the location of the start of the growing DNA loop.

Extended Data Fig. 3 Estimation of condensin-binding position on supercoiled DNA measured with AFM.

a) Representative AFM images showing condensin binding near a plectoneme body (left) and near the plectoneme tip (right). Data represent 4 independent experiments. b) Statistics showing enrichment of condensin as observed outside of plectoneme, at plectoneme body, and at apical loops. In order to avoid possible supercoiling induced by condensin, the plasmids were incubated with AMP-PNP.

Source data

Extended Data Fig. 4 Additional examples showing that condensin initially loads near a plectoneme tip and moves downwards during plectoneme loop extrusion.

a) Snapshots showing condensin moving from the plectoneme tip (1st) towards the stem (till 3rd snapshot). Simultaneously the plectoneme becomes compacted and moves together with the condensin during extrusion while it grows back its length at later times (3rd – 5th frame). Data represent 3 independent experiments. b) Snapshot (left) and kymograph (right) showing condensin moving from the plectoneme tip towards the stem. Data represent 3 independent experiments. (c) Change in position for condensins that bound at the tip and moved along the length of DNA plectonemes extracted from the kymographs as in panel b for N = 6 molecules. The red colored trace corresponds to the kymograph in panel b.

Extended Data Fig. 5 Condensin-mediated loop extrusion on supercoiled DNA is strictly asymmetric.

(a) Examples of the DNA length calculated from the integrated fluorescence intensity kymographs of DNA loop extrusion by a single condensin on supercoiled DNA, showing asymmetric loop extrusion. (b) Same for an event where multiple condensins underlie the DNA loop, showing symmetric loop extrusion. (c) Statistics showing the ratios of the asymmetric and symmetric loop extrusion events observed for a single condensin (1) and multiple condensins (1 + ).

Source data

Extended Data Fig. 6 While plectonemes connected to the anchor side of condensin disappear, the corresponding DNA amount does not decrease.

(a) Example kymograph (top) and the DNA peaks (bottom) detected during condensin-mediated loop extrusion on supercoiled DNA. (b) Corresponding DNA lengths calculated from the kymograph. During the loop growth (blue; 45 s- 60 s) the length of DNA connected to the anchor side of condensin (pink) did not decrease, but instead DNA was accumulated from the motor side of condensin (green). This indicates that the disappearance of plectoneme at the anchor side is not due to the absorbance of plectoneme into the loop but rather to the stretching of the DNA.

Extended Data Fig. 7 Additional examples of condensin location along the plectoneme.

Condensin was found to be located at (a) the stem or (b) the middle after extrusion of a supercoiled loop in the case of single condensin. (c) Additional examples where multiple condensins are located along the supercoiled loop. Data in (a-c) represent 5 independent experiments.

Extended Data Fig. 8 AFM analysis of supercoiled DNA molecules.

(a) AFM image of a non-entangled dsDNA molecule. (b) Same image in an expanded blue-red color scale to highlight the entangled regions of dsDNA (red) versus the non-entangled regions (light blue) and the mica (dark blue). Inset shows the Z color scale. Data in (a,b) represent 3 independent experiments. (c) Height profile along the molecule in a/b, showing an average height of 1.25 nm. (d) AFM image of a dsDNA molecule with one crossover. (e) Same image in an expanded blue-red color scale. The crossover point is denoted with a yellow arrow. Data in (d,e) represent 5 independent experiments. (f) Height profile along the molecule shown on d/e. A locally increased height of > 2 nm is observed at the crossing point (arrows). The arrows denote the two peaks that correspond to the crossing point – which is encountered twice upon tracking the contour of the DNA along this ‘figure-8’ profile. (g) AFM image of entangled dsDNA molecules. Yellow arrows signal the molecules of which the profiles are shown in panels h and i. Data represent 3 independent experiments. (h) Height profile along a molecule where the entangled patches are denoted in red. (i) Same for the molecule indicated by i in panel g. Notably, this molecule was almost entirely entangled along its full contour length. Scale bars are 200 nm.

Extended Data Fig. 9 Examples of bleaching time traces of fluorescence intensities of individual ATTO647N-labelled condensin complexes at the location of supercoiled loops.

(a) Bleaching occurred in a single step-wise manner for condensin after a loop extrusion event. (b) Bleaching occurred in a double step-wise manner after an event where two condensins had merged into a single supercoiled DNA loop. (c) Bleaching occurred in a single step-wise manner for condensin after a loop extrusion event. Additional short binding events occurred at the plectoneme loop.

Extended Data Fig. 10 Condensin binding is length and supercoiling dependent.

(a) Observation probability of condensin on nicked DNA with two different lengths (21 kb and 48.5 kb), estimated by summing the total number of detected condensin peaks over all time points, and dividing this by the DNA length and measurement time. N = 8 molecules for 21 kb, N = 22 molecules for 48 kb length, respectively. (b) Condensin enrichment on supercoiled loops and relaxed loops, estimated by summing up the total intensity of the detected condensin peaks within the loop region with similar lengths (~60 % of its contour length). N = 25 molecules for supercoiled loops and N = 20 for relaxed loops, respectively. Data represent mean ± standard deviation.

Source data

Supplementary information

Supplementary Information

Legends for Supplementary Videos 1 and 2.

Reporting Summary

Peer Review File

Supplementary Video 1

Video showing condensin-mediated DNA loop extrusion on supercoiled DNA. A single DNA intensity peak appeared among the multiple plectoneme peaks upon loop extrusion, which gradually increased in size as it moved along the DNA.

Supplementary Video 2

Video showing that condensin initially bound to a plectoneme gradually reels in DNA while simultaneously localizing all of the nearby plectonemes. After the loop was formed, we applied a buffer flow and monitored the location of the condensin along the supercoiled DNA loop. This revealed that condensin was localized at the stem of the plectonemic loop.

Source data

Source Data Fig. 1

Statistical source data of Fig. 1e,g,h.

Source Data Fig. 2

Statistical source data of Fig. 2c,e.

Source Data Fig. 3

Statistical source data of Fig. 3g.

Source Data Fig. 4

Statistical source data of Fig. 4a,b.

Source Data Fig. 5

Statistical source data of Fig. 5b–d.

Source Data Fig. 6

Statistical source data of Fig. 6g,h.

Source Data Extended Data Fig. 1

Statistical source data of Extended Data Fig. 1.

Source Data Extended Data Fig. 3

Statistical source data of Extended Data Fig. 3b.

Source Data Extended Data Fig. 5

Statistical source data of Extended Data Fig. 5c.

Source Data Extended Data Fig. 10

Statistical source data of Extended Data Fig. 10a,b.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, E., Gonzalez, A.M., Pradhan, B. et al. Condensin-driven loop extrusion on supercoiled DNA. Nat Struct Mol Biol 29, 719–727 (2022). https://doi.org/10.1038/s41594-022-00802-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-022-00802-x

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation