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PICH and TOP3A cooperate to induce positive DNA supercoiling

Abstract

All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.

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Data availability

Source data for Figs. 4d,e, 5e and 7e are available with the paper online. Other datasets and materials generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Code availability

The custom-made program used to operate the tweezers and analyze the data can be obtained upon request to V. Croquette (ENS Paris).

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

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Acknowledgements

We thank R. Singh Thakur for purification of recombinant proteins, and members of the Hickson group for helpful discussions. We also thank E. Hoffmann, H. Mankouri and C. Nielsen for helpful comments on the manuscript, S. Kowalczykowski (University of California at Davis, USA) for purified E. coli Top3 and M. T. Kanemaki (National Institute of Genetics, Mishima, Shizuoka, Japan) for the degron cells. This work was supported by the Danish National Research Foundation (No. DNRF115, to I.D.H.), the European Union Horizon 2020 ‘Chromavision’ (No. 665233, to I.D.H.) and the Nordea Foundation (to I.D.H.). T. Hassenkam wishes to thank the Villum foundation “Experiment” for support.

Author information

A.H.B. performed the experiments. A.H.B. and I.D.H. designed the experiments and analyzed the data. J.-F.A. helped carry out the experiments with magnetic tweezers and data analysis. T.H. assisted with A.F.M.’s experiments. M.P. assisted with cell biology experiments. K.S. and M.I.S. assisted with ensemble biochemistry experiments. I.D.H. supervised the work. A.H.B. and I.D.H. wrote the manuscript, and all authors edited it.

Competing interests

The authors declare no competing interests.

Correspondence to Anna H. Bizard or Ian D. Hickson.

Integrated supplementary information

  1. Supplementary Figure 1 PICH cooperates with TRR to induce DNA positive supercoiling.

    (ac) Representative 1D agarose gel electrophoresis showing the influence of increasing concentrations of PICH on the topoisomerase activity catalyzed by hTop1 (a), hTop2a (b), and TRR (c) using a negatively supercoiled plasmid as a substrate. Agarose gel electrophoresis was either performed in the absence (neutral; a, b) or in the presence of chloroquine (c) in order to reveal variations of topology within the negatively supercoiled topoisomers. For each experiment, relaxed (rel), negatively (-) moderately positively (rel+), and positively (+) supercoiled plasmids were used as markers. (d) Representative 2D agarose gel electrophoresis of the reaction products shown in (c). For each panel, representative images of at least 3 independent experiments are presented. Each independent experiment lead to similar results.

  2. Supplementary Figure 2 PICH and TRR introduce a high density of positive supercoiling.

    Representative AFM topographs of the relaxed substrate (a), a positively supercoiled (+8) marker (b), and the PICH-TRR reaction product (c). For each example, a cartoon depicting the conformation of two selected molecules is presented. For each panel, images representative of at least 20 images collected for 3 independent experiments are presented. All experiments lead to similar results. (d) Additional examples of representative AFM topographs of several PICH-TRR reaction products. For each topological form, a zoomed image is presented. White arrowheads in the zoomed panels indicate the presence of height peaks. White numbers indicate the amount of positive supercoils in the corresponding field of view as estimated by counting of the height peaks. Height scale bar is common for all images, and is shown on the right. XY scale bar is 100 nm.

  3. Supplementary Figure 3 Evidence that supercoiling occurs via a translocation-dependent mechanism.

    (a, b) Schematic representation of mechanistic models for PICH-mediated positive supercoiling. PICH is depicted as a red circle. Positive and negative torsional stress induced by PICH translocation are indicated by red “+” and “-” symbols (Translocation models; b). The (-) and (+) symbols indicate the topology of DNA segments resulting from PICH wrapping DNA (Wrapping model; a) or from PICH translocation (Translocation model; b). Net supercoiling level (ΔLk) of each intermediate is indicated in the gray bar below each panel. (a) In the wrapping model, the bound DNA segment might be wrapped around PICH in a positive supercoiling conformation, leading to the introduction of compensatory negative supercoils in the unbound DNA segment. Type IA (upper panel) and Type IB or II topoisomerases (lower panel) then relax the negative supercoils, resulting into the introduction of net positive supercoiling. (b) In the translocation model, and according to the twin-supercoiling domain model, PICH translocation might lead to the redistribution of DNA torsional stress leading to the accumulation of unconstrained positive and negative supercoiling, respectively, in front and behind PICH. Type IA (upper panel) would only relax the negative supercoils, resulting in the introduction of net positive supercoiling. Type IB or II topoisomerases (lower panel) would relax both positive and negative supercoils, leading to no net alteration in supercoiling. A topological barrier induced by PICH is depicted as a black line and red square. (c-f) Representative 1D agarose gel electrophoresis showing the influence of PICH on the activity catalyzed by increasing concentrations of wgTop1 (c), ecTop1 (d), ecTop3 (e), and TRR (f) using a negatively supercoiled plasmid as a substrate. Agarose gel electrophoresis was performed in the absence (neutral) (c, d) or in the presence of chloroquine (e, f) in order to reveal variations of topology within the negatively supercoiled topoisomers. For each experiment, open circular (oc), linear (lin), relaxed (rel), negatively (-) and positively (+) supercoiled plasmids were used as markers. For each panel, representative images out of at least 3 independent experiments are presented. Each independent experiment lead to similar results. (g) Additional examples of representative AFM topographs of open circular DNA plasmids incubated in the presence of PICH and ATPγS. Height scale bar is common for all images and is shown on the right. XY scale bar is 100 nm. Images representative of at least 10 images collected for 3 independent experiments are presented. All experiments lead to similar results.

  4. Supplementary Figure 4 Evidence that PICH exhibits a DNA loop extrusion activity and induces redistribution of DNA torsional stress.

    (a) Cartoon summarizing the features of the magnetic tweezer setup. (b) Additional representative traces showing the variation of DNA extension over time observed in the presence of PICH and ATP, at a force of 2 pN and on nicked DNA molecules. (c) Histogram to indicate the processivity of each event observed on nicked DNA molecules and at 2 pN (n = 273 events; bin size = 10 nm; errors bars are statistical errors; the red curve correspond to an exponential fit. (d) Representative traces showing the variation of DNA extension over time observed in the presence of PICH and ATP on nicked DNA molecules at various forces; 0.5 pN, 2 pN or 8 pN. (e) Scan showing the influence of rotation of the magnets on the extension of a nicked (gray curve) and a coilable (purple curve) DNA molecule at a force of 2 pN. (f) Additional representative traces showing the variation of DNA extension over time observed in the presence of PICH and ATP, at a force of 2 pN, on coiled (+ 100) DNA molecules.

  5. Supplementary Figure 5 TRR facilitates decatenation of UFBs by Top2a.

    (a) Western blot analysis of Top3a, PICH and Top2a in HCT116-Top3aAID (Top3dg) and in the control cell line (430) in metaphase arrested cells incubated for 3 h in the absence (-) or in the presence (+) of Auxin. For each panel, representative images out of at least 3 independent experiments are presented. Uncropped blots images are shown in Supplementary Data Set 1. (b) Quantification of the average number of UFBs in the control cell line (430) exposed (red bars) or not (gray bars) to Auxin. Cells were treated with solvent (-) or either 50 nM or 100 nM ICRF193, as indicated below. Error bars denote the standard deviation of 3 independent replicates (n = 80cells/replicate) (c) Additional representative immunofluorescence images of HCT116-Top3aAID cells following Top3a depletion (+Auxin) in cells progressing through anaphase in the absence of ICRF193. UFBs are marked by PICH (blue) and centromeres are marked by CREST (red). The zoomed image shows CREST-positive staining at the tips of UFBs. Scale bar is 10 µm. Representative images of at least 20 images collected for 3 independent replicates are presented.

Supplementary information

  1. Supplementary Figures and Supplementary Dataset

    Supplementary Figures 1–5 and Supplementary Dataset 1

  2. Reporting Summary

Source data

  1. Source Data Fig. 4d,e

  2. Source Data Fig. 5e

  3. Source Data Fig. 7e

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Fig. 1: PICH cooperates with TRR to induce positive DNA supercoiling.
Fig. 2: PICH and TRR introduce a high density of positive supercoiling.
Fig. 3: Evidence that supercoiling occurs via a translocation-dependent mechanism.
Fig. 4: Evidence that PICH exhibits a DNA loop–extrusion activity.
Fig. 5: Evidence that PICH induces DNA torsional stress redistribution.
Fig. 6: Model for the proposed catalytic cycle.
Fig. 7: TRR facilitates UFB decatenation by Top2a.
Supplementary Figure 1: PICH cooperates with TRR to induce DNA positive supercoiling.
Supplementary Figure 2: PICH and TRR introduce a high density of positive supercoiling.
Supplementary Figure 3: Evidence that supercoiling occurs via a translocation-dependent mechanism.
Supplementary Figure 4: Evidence that PICH exhibits a DNA loop extrusion activity and induces redistribution of DNA torsional stress.
Supplementary Figure 5: TRR facilitates decatenation of UFBs by Top2a.