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Cohesin regulates homology search during recombinational DNA repair

Nature Cell Biology volume 23, pages 1176–1186 (2021)Cite this article

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Abstract

Homologous recombination repairs DNA double-strand breaks (DSB) using an intact dsDNA molecule as a template. It entails a homology search step, carried out along a conserved RecA/Rad51-ssDNA filament assembled on each DSB end. Whether, how and to what extent a DSB impacts chromatin folding, and how this (re)organization in turns influences the homology search process, remain ill-defined. Here we characterize two layers of spatial chromatin reorganization following DSB formation in Saccharomyces cerevisiae. Although cohesin folds chromosomes into cohesive arrays of ~20-kb-long chromatin loops as cells arrest in G2/M, the DSB-flanking regions interact locally in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin, Mec1ATR, Rad52 and Rad51. This local structure blocks cohesin progression, constraining the DSB region at the base of a loop. Functionally, cohesin promotes DSB–dsDNA interactions and donor identification in cis, while inhibiting them in trans. This study identifies multiple direct and indirect ways by which cohesin regulates homology search during recombinational DNA repair.

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Fig. 1: HR repair takes place in the context of individualized chromosomes structured by cohesins as arrays of chromatin loops.
Fig. 2: Tethering interaction across the DSB region is resection- and 9-1-1 clamp-dependent.
Fig. 3: The DSB region is a cohesin translocation roadblock.
Fig. 4: The 9-1-1 clamp blocks cohesin translocation, which traps the DSB region at the loop base.
Fig. 5: Cohesins promote identification of nearby over inter-chromosomal homologies.
Fig. 6: Cohesin promotes homology identification in cis and inhibits it in trans in multiple ways.

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Data availability

Sequencing datasets have been deposited in the Sequence Read Archive under accession code PRJNA647790. The GEO accession number for the processed data is GSE179646. The reference genome for S. cerevisiae W303 is provided at https://www.ncbi.nlm.nih.gov/assembly/GCA_002163515.1/ and for C. glabrata at http://www.candidagenome.org/download/sequence/C_glabrata_CBS138/current/. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Open-access versions of the programs and pipeline used (Hicstuff78, Chromosight30 and Serpentine45) are available online on GitHub at https://github.com/koszullab/, as well as HiCstuff78 (www.github.com/koszullab/hicstuff) version 3.0.1, Chromosight30 (www.github.com/koszullab/chromosight) version 1.4.1, Serpentine45 (www.github.com/koszullab/serpentine) version 0.1.3, FlowJo software 10.8.0, MACS282 (version 2.2.6 available online at https://pypi.org/project/MACS2/), R83 (version x64 3.6.2 is available online at https://cloud.r-project.org/), Bowtie284 (version 2.3.4.1 available online at http://bowtie-bio.sourceforge.net/bowtie2/), SAMtools85 (version 1.9 available online at http://www.htslib.org/download/http://www.htslib.org/download/), Bedtools86 (version 2.29.1 available online at https://bedtools.readthedocs.io/en/latest/content/installation.html) and Cooler81 (versions 0.8.7–0.8.11 available online at https://cooler.readthedocs.io/en/latest/).

References

  1. Renkawitz, J., Lademann, C. A. & Jentsch, S. Mechanisms and principles of homology search during recombination. Nat. Rev. Mol. Cell Biol. 15, 369–383 (2014).

    CAS  PubMed  Google Scholar 

  2. Bell, J. C. & Kowalczykowski, S. C. RecA: regulation and mechanism of a molecular search engine. Trends Biochem. Sci. 41, 491–507 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Forget, A. L. & Kowalczykowski, S. C. Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. Nature 482, 423–427 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Yang, H., Zhou, C., Dhar, A. & Pavletich, N. P. Mechanism of strand exchange from RecA-DNA synaptic and D-loop structures. Nature 586, 801–806 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Burgess, S. M. & Kleckner, N. Collisions between yeast chromosomal loci in vivo are governed by three layers of organization. Genes Dev. 13, 1871–1883 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Agmon, N., Liefshitz, B., Zimmer, C., Fabre, E. & Kupiec, M. Effect of nuclear architecture on the efficiency of double-strand break repair. Nat. Cell Biol. 15, 694–699 (2013).

    CAS  PubMed  Google Scholar 

  7. Lee, C.-S. et al. Chromosome position determines the success of double-strand break repair. Proc. Natl Acad. Sci. USA 113, E146–E154 (2016).

    CAS  PubMed  Google Scholar 

  8. Batté, A. et al. Recombination at subtelomeres is regulated by physical distance, double-strand break resection and chromatin status. EMBO J. 36, 2609–2625 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. Arnould, C. & Legube, G. The secret life of chromosome loops upon DNA double-strand break. J. Mol. Biol. 432, 724–736 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sanders, J. T. et al. Radiation-induced DNA damage and repair effects on 3D genome organization. Nat. Commun. 11, 6178 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Waterman, D. P., Haber, J. E. & Smolka, M. B. Checkpoint responses to DNA double-strand breaks. Annu. Rev. Biochem. 89, 103–133 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Arnould, C. et al. Loop extrusion as a mechanism for formation of DNA damage repair foci. Nature 590, 660–665 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ström, L., Lindroos, H. B., Shirahige, K. & Sjögren, C. Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. Mol. Cell 16, 1003–1015 (2004).

    PubMed  Google Scholar 

  14. Heidinger-Pauli, J. M., Unal, E., Guacci, V. & Koshland, D. The kleisin subunit of cohesin dictates damage-induced cohesion. Mol. Cell 31, 47–56 (2008).

    CAS  PubMed  Google Scholar 

  15. Ünal, E. et al. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol. Cell 16, 991–1002 (2004).

    PubMed  Google Scholar 

  16. Ünal, E., Heidinger-Pauli, J. M. & Koshland, D. DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7). Science 317, 245–248 (2007).

    PubMed  Google Scholar 

  17. Ünal, E. et al. A molecular determinant for the establishment of sister chromatid cohesion. Science 321, 566–569 (2008).

    PubMed  Google Scholar 

  18. Kadyk, L. C. & Hartwell, L. H. Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae. Genetics 132, 387–402 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Birkenbihl, R. P. & Subramani, S. Cloning and characterization of rad21 an essential gene of Schizosaccharomyces pombe involved in DNA double-strand-break repair. Nucleic Acids Res. 20, 6605–6611 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Sjögren, C. & Nasmyth, K. Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae. Curr. Biol. 11, 991–995 (2001).

    PubMed  Google Scholar 

  21. Covo, S., Westmoreland, J. W., Gordenin, D. A. & Resnick, M. A. Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes. PLoS Genet. 6, e1001006 (2010).

    PubMed  PubMed Central  Google Scholar 

  22. Gelot, C. et al. The Cohesin Complex Prevents the End Joining of Distant DNA Double-Strand Ends. Mol. Cell 61, 15–26 (2016).

    CAS  PubMed  Google Scholar 

  23. Cheblal, A. et al. DNA damage-induced nucleosome depletion enhances homology search independently of local break movement. Mol. Cell 80, 311–326 (2020).

    CAS  PubMed  Google Scholar 

  24. Dion, V., Kalck, V., Seeber, A., Schleker, T. & Gasser, S. M. Cohesin and the nucleolus constrain the mobility of spontaneous repair foci. EMBO Rep. 14, 984–991 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Schalbetter, S. A. et al. Structural maintenance of chromosome complexes differentially compact mitotic chromosomes according to genomic context. Nat. Cell Biol. 19, 1071–1080 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Lazar‐Stefanita, L. et al. Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle. EMBO J. 36, 2684–2697 (2017).

    PubMed  PubMed Central  Google Scholar 

  27. Garcia-Luis, J. et al. FACT mediates cohesin function on chromatin. Nat. Struct. Mol. Biol. 26, 970–979 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Dauban, L. et al. Regulation of cohesin-mediated chromosome folding by Eco1 and other partners. Mol. Cell 77, 1279–1293 (2020).

    CAS  PubMed  Google Scholar 

  29. Piazza, A. et al. Dynamic processing of displacement loops during recombinational DNA repair. Mol. Cell 73, 1255–1266 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Matthey-Doret, C. et al. Computer vision for pattern detection in chromosome contact maps. Nat. Commun. 11, 5795 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Paldi, F. et al. Convergent genes shape budding yeast pericentromeres. Nature 582, 119–123 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Costantino, L., Hsieh, T.-H. S., Lamothe, R., Darzacq, X. & Koshland, D. Cohesin residency determines chromatin loop patterns. eLife 9, e59889 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Tercero, J. A., Labib, K. & Diffley, J. F. X. DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p. EMBO J. 19, 2082–2093 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Mitter, M. et al. Conformation of sister chromatids in the replicated human genome. Nature 586, 139–144 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Symington, L. S. Mechanism and regulation of DNA end resection in eukaryotes. Crit. Rev. Biochem. Mol. Biol. 51, 195–212 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhu, Z., Chung, W.-H., Shim, E. Y., Lee, S. E. & Ira, G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double strand break ends. Cell 134, 981–994 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Mimitou, E. P. & Symington, L. S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 (2008).

    CAS  PubMed  Google Scholar 

  38. Bantele, S. C. S., Lisby, M. & Pfander, B. Quantitative sensing and signalling of single-stranded DNA during the DNA damage response. Nat. Commun. 10, 944 (2019).

    PubMed  PubMed Central  Google Scholar 

  39. Nakai, W., Westmoreland, J., Yeh, E., Bloom, K. & Resnick, M. A. Chromosome integrity at a double-strand break requires exonuclease 1 and MRX. DNA Repair 10, 102–110 (2011).

    CAS  PubMed  Google Scholar 

  40. Lisby, M. & Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713 (2004).

    CAS  PubMed  Google Scholar 

  41. Zhang, H. et al. Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p. J. Biol. Chem. 276, 26715–26723 (2001).

    CAS  PubMed  Google Scholar 

  42. Chan, K.-L. et al. Pds5 promotes and protects cohesin acetylation. Proc. Natl Acad. Sci. USA 110, 13020–13025 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Murayama, Y. & Uhlmann, F. DNA entry into and exit out of the cohesin ring by an interlocking gate mechanism. Cell 163, 1628–1640 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Fasching, C. L., Cejka, P., Kowalczykowski, S. C. & Heyer, W.-D. Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism. Mol. Cell 57, 595–606 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Baudry, L., Millot, G. A., Thierry, A., Koszul, R. & Scolari, V. F. Serpentine: a flexible 2D binning method for differential Hi-C analysis. Bioinformatics 36, 3645–3651 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wright, W. D., Shah, S. S. & Heyer, W.-D. Homologous recombination and the repair of DNA double-strand breaks. J. Biol. Chem. 293, 10524–10535 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Coïc, E. et al. Dynamics of homology searching during gene conversion in Saccharomyces cerevisiae revealed by donor competition. Genetics 189, 1225–1233 (2011).

    PubMed  PubMed Central  Google Scholar 

  48. von Hippel, P. H. & Berg, O. G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989).

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Vian, L. et al. The energetics and physiological impact of cohesin extrusion. Cell 173, 1165–1178 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Dai, H.-Q. et al. Loop extrusion mediates physiological Igh locus contraction for RAG scanning. Nature 590, 338–343 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Ba, Z. et al. CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning. Nature 586, 305–310 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Li, K., Bronk, G., Kondev, J. & Haber, J. E. Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break. Proc. Natl Acad. Sci. USA 117, 21354–21363 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Koszul, R., Caburet, S., Dujon, B. & Fischer, G. Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments. EMBO J. 23, 234–243 (2004).

    CAS  PubMed  Google Scholar 

  56. Koszul, R., Dujon, B. & Fischer, G. Stability of large segmental duplications in the yeast genome. Genetics 172, 2211–2222 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Gresham, D. et al. Adaptation to diverse nitrogen-limited environments by deletion or extrachromosomal element formation of the GAP1 locus. Proc. Natl Acad. Sci. USA 107, 18551–18556 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Savocco, J. & Piazza, A. Recombination-mediated genome rearrangements. Curr. Opin. Genet. Dev. 71, 63–71 (2021).

    CAS  PubMed  Google Scholar 

  59. Hull, R. M. et al. Transcription-induced formation of extrachromosomal DNA during yeast ageing. PLoS Biol. 17, e3000471 (2019).

    PubMed  PubMed Central  Google Scholar 

  60. Dennis, M. Y. & Eichler, E. E. Human adaptation and evolution by segmental duplication. Curr. Opin. Genet. Dev. 41, 44–52 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Wilde, J. J. et al. Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair. Cell 184, 3267–3280 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Du, Z. et al. Allelic reprogramming of 3D chromatin architecture during early mammalian development. Nature 547, 232–235 (2017).

    CAS  PubMed  Google Scholar 

  63. Ke, Y. et al. 3D chromatin structures of mature gametes and structural reprogramming during mammalian embryogenesis. Cell 170, 367–381 (2017).

    CAS  PubMed  Google Scholar 

  64. Gassler, J. et al. A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture. EMBO J. 36, 3600–3618 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Zickler, D. & Kleckner, N. Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33, 603–754 (1999).

    CAS  PubMed  Google Scholar 

  66. Muller, H. et al. Characterizing meiotic chromosomes’ structure and pairing using a designer sequence optimized for Hi-C. Mol. Syst. Biol. 14, e8293 (2018).

    PubMed  PubMed Central  Google Scholar 

  67. Schalbetter, S. A., Fudenberg, G., Baxter, J., Pollard, K. S. & Neale, M. J. Principles of meiotic chromosome assembly revealed in S. cerevisiae. Nat. Commun. 10, 4795 (2019).

    PubMed  PubMed Central  Google Scholar 

  68. Kim, K. P. et al. Sister cohesion and structural axis components mediate homolog bias of meiotic recombination. Cell 143, 924–937 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Lam, I. & Keeney, S. Mechanism and regulation of meiotic recombination initiation. Cold Spring Harb. Perspect. Biol. 7, a016634 (2015).

    PubMed Central  Google Scholar 

  70. Brown, M. S., Grubb, J., Zhang, A., Rust, M. J. & Bishop, D. K. Small Rad51 and Dmc1 complexes often co-occupy both ends of a meiotic DNA double strand break. PLoS Genet. 11, e1005653 (2015).

    PubMed  PubMed Central  Google Scholar 

  71. Panizza, S. et al. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination. Cell 146, 372–383 (2011).

    CAS  PubMed  Google Scholar 

  72. Hunter, N. & Kleckner, N. The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell 106, 59–70 (2001).

    CAS  PubMed  Google Scholar 

  73. Piazza, A., Rajput, P. & Heyer, W.-D. in Homologous Recombination: Methods and Protocols Vol. 2153 (eds Aguilera, A. & Carreira, A.) 535–554 (Springer, 2021).

  74. Dauban, L. et al. Quantification of the dynamic behaviour of ribosomal DNA genes and nucleolus during yeast Saccharomyces cerevisiae cell cycle. J. Struct. Biol. 208, 152–164 (2019).

    CAS  PubMed  Google Scholar 

  75. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast Chichester Engl. 14, 953–961 (1998).

    CAS  Google Scholar 

  76. Hu, B. et al. Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq. Nucleic Acids Res. 43, e132 (2015).

    PubMed  PubMed Central  Google Scholar 

  77. Petela, N. J. et al. Scc2 is a potent activator of cohesin’s ATPase that promotes loading by binding Scc1 without Pds5. Mol. Cell 70, 1134–1148 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Cyril M.-D. et al. koszullab/hicstuff: use miniconda layer for docker and improved P(s) normalisation. https://doi.org/10.5281/zenodo.4066363 (Zenodo, 2020).

  79. Cournac, A., Marie-Nelly, H., Marbouty, M., Koszul, R. & Mozziconacci, J. Normalization of a chromosomal contact map. BMC Genomics 13, 436 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Imakaev, M. et al. Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9, 999–1003 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Abdennur, N. & Mirny, L. A. Cooler: scalable storage for Hi-C data and other genomically labeled arrays. Bioinformatics 36, 311–316 (2020).

    CAS  PubMed  Google Scholar 

  82. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

  83. R Core Team. R: a language and environment for statistical computing (R Foundation for Statistical Computing); https://www.R-project.org/ (2017).

  84. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  86. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank A. Cournac, C. Matthey-Doret, V. Scolari, J. Serizay, G. Millot and T. Foutel-Rodier for their help with bioinformatics analysis and sharing unpublished scripts and programs, C. Chapard for his help with cell synchronization, ChIP-seq and for providing test Hi-C datasets, as well as all members of the Koszul laboratory and of the Parisian Yeast club for stimulating discussions. We thank F. Beckouët for giving us the C. glabrata strains for ChIP-seq calibration and the FB09-9C/yLD143-1a strains bearing the Scc1-Pk9/6HA constructs. We thank K. Dubrana for giving us Longtine vectors. We thank Z. Xu for giving us the yT598 strain bearing the mec1 sml1 deletions. Finally, we are grateful to N. Kleckner, W.-D. Heyer, S. Gasser, B. Llorente, G. Liti, K. Dubrana and D. Leach for helpful discussions and comments on the manuscript. This research was supported by the CNRS as part of its Momentum programme to A.P., and the European Research Council (ERC) under the European Union’s Horizon 2020 programme to A.P. and R.K. (ERC grants nos. 851006 and 771813, respectively).

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Author notes

  1. These authors contributed equally: Aurèle Piazza, Hélène Bordelet.

Authors and Affiliations

  1. Institut Pasteur, CNRS UMR3525, Unité Régulation Spatiale des Génomes, F-75015, Paris, France

    Aurèle Piazza, Hélène Bordelet, Agnès Thierry, Fabien Girard & Romain Koszul

  2. Université de Lyon, ENS de Lyon, Université Claude Bernard, Laboratoire de Biologie et Modélisation de la Cellule, CNRS UMR5239, INSERM U1210, 46 allée d’Italie, 69007, Lyon, France

    Aurèle Piazza, Hélène Bordelet, Agnès Dumont & Jérôme Savocco

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Contributions

Conceptualization was provided by A.P. and R.K. Experiments were conducted by A.P., H.B., A.D., F.G. and A.T. Data analysis was carried out by A.P., H.B. and J.S. The data were interpreted by A.P., H.B. and R.K. Supervision was provided by A.P. and R.K. Funding acquisition was carried out by A.P. and R.K. The original draft of the manuscript was written by A.P. and H.B. Subsequent and final drafts were produced by A.P., H.B. and R.K.

Corresponding authors

Correspondence to Aurèle Piazza or Romain Koszul.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks Irene Chiolo, Leonid Mirny and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Genome-wide spatial chromatin reorganization following DSB formation in wild-type cells.

a Hi-C coverage binned at 5 kb prior to and following DSB induction, normalized onto the average coverage in absence of DSB. Data represent the mean of a biological replicate, except for DSB 2 hrs (experiment done once). b Cell-cycle progression prior to and following DSB induction determined by flow cytometry. The percentage of cells with a 2n DNA content is indicated for each time point.c Top left: contact map showing intra- and inter-chromosomal contacts made by chromosomes II, IV, V, XIII in a wild-type strain (APY295) 4 hours post-DSB induction. White and black arrows: centromeres and telomeres contacts, respectively. The Rabl chromosome organization is maintained (clustered centromeres, enriched telomere contacts). Bottom right: contact maps ratio obtained in a wt strain 4 hours post-DSB induction (red) over asynchronous cells without a DSB (blue). Bin size: 5 kb. Representative biological replicate. d Heatmap of loop lengths and scores in wt cells either asynchronous, G1-arrested, or metaphase-arrested without a DSB, or at 4 hours post-DSB induction. n: number of called loops. Datasets subsampled to 24 million contacts each. e Aggregated ratio maps of 29 kb windows centered on centromeres over randomly chosen windows, determined from data presented in Fig. 1c. An undamaged G1 control is shown for comparison. Bin size: 1 kb. f Contact maps of regions of chr. V and IV in strains bearing either no HOcs (n=1), Chr. V-HOcs (n=2) or Chr. IV-HOcs (n=2) 4 hours post-induction of HO (APY143, APY295, and APY526). Red dot: DSB position (and LIP). Subsampling to 24 million contacts each. Bin size: 1 kb. g Contact probability as a function of genomic distance (Pc(s), left) and its derivative (right) determined from samples in (f). h Contact maps of a region of Chr. V before and at 2, 4, and 6 hours post-DSB induction (n=3, 3, 3, and 1 biological replicates, respectively) using a Hi-C protocol with a single DpnII digestion. Generated from 17-19 million contacts each. Red dot: DSB position. White dot: centromere. Bin size: 2 kb. i Hi-C coverage binned at 5 kb in samples in (h) as in Extended Data Fig. 1a. Data represents mean ±SD. n=3 for all samples, except DSB 6 hr (n=1). Vertical dotted line: position of the DSB. j Contact probability as a function of genomic distance (Pc(s), left) and its derivative (right) determined from replicates of samples in (h).

Source data

Extended Data Fig. 2 Spatial genome reorganization upon formation of a repairable DSB in diploid cells.

a Contact maps of a region of Chr. V in diploid wild-type cells (APY531) prior to and 4 hours post-induction of a heterozygous DSB (Red dot). Generated from 17 million contacts each. White dot: centromere. Bin size: 1 kb. b Hi-C coverage binned at 5 kb before and at 2, 4, and 6 hours post-DSB induction as in Extended Data Fig. 1a (n=1 experiment each). Coverage drops at 2 hours and is progressively restored at 4 and 6 hours, as inter-homolog repair occurs5. The minimum coverage expected for a heterozygous DSB is 0.5 (horizontal dotted line). Vertical dotted line: position of the DSB. Horizontal dotted line at 1: coverage of the undamaged control. c Derivative of the contact probability as a function of genomic distance Pc(s) before and at 2, 4, and 6 hours post-DSB induction. d Proportion of intra-chromosomal vs. total contacts for each 16 chromosomes in diploid wild-type cells before and at 2, 4, and 6 hours post-DSB induction (n=1 experiment each). Red bar: median.

Extended Data Fig. 3 Spatial genome reorganization upon formation of a DSB in G1 blocked or released cells.

a Experimental scheme to undergo DSB induction in G1 released or G1 blocked cells (APY331 and APY535, respectively). b Cell-cycle progression for damaged G1 blocked cells: prior to alpha factor addition (-3h), 3 hours later (0h), and 4 hours after galactose addition. FACS analysis for G1 released cells are presented in Extended Data Fig. 4i. c Left : Hi-C coverage, same as in Extended Data Fig. 1a., binned at 5 kb of samples in (d): G1 arrested cells without DSB, and G1-arrested and G1-released cells 4 hours after DSB induction (n = 1, 1, and 2 biological replicates, respectively). Right : DSB efficiency measured by relative qPCR of the HO site compared to an undamaged locus (see Methods). Vertical dotted line: position of the DSB. Horizontal dotted line: coverage of the undamaged control. d Contact maps of chr. V undamaged (left) and 4 hours after DSB induction in G1-arrested (middle) or -released (right) cells (APY266, APY535 and APY331 respectively). Bin size = 1 kb. e Contact probability as a function of genomic distance (Pc(s), left) and its derivative (right) determined from samples in (d). f Proportion of intra-chromosomal vs. total contacts for each 16 chromosomes determined from samples in (d). Red bar: median.

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Extended Data Fig. 4 Loop formation and chromosome individualization following DSB formation is Cohesin-dependent and independent of replication and the sister chromatid.

a Left panels: contact maps of a region of Chr. V in untagged or Scc1-AID tagged cells at 4 hours post-DSB (APY466 and APY467, respectively), treated with DMSO added at the same time as DSB induction. Bin size: 1 kb. Bottom left: ratio map of Scc1-AID tagged over untagged cells, treated with DMSO at 4 hours post DSB. The AID tag mildly alters cohesin regulation, resulting in longer loops. Blue: stronger signal in untagged cells; vice-versa for red. Right panels: contact maps of a region of Chr. V in untagged or Scc1-AID tagged cells 4 hours post-DSB induction, treated with IAA added 2 hours after DSB induction (Fig. 1e and Extended Data Fig. 1). Bin size: 1 kb. Bottom right: ratio map of Scc1-AID tagged over untagged cells treated with IAA (2hrs.). b Western blot of Scc1-(Pk3-)AID probed with an anti-Pk antibody (top) and a loading control GAPDH (bottom) over the course of IAA or DMSO (mock) treatment of samples presented in (a) and Fig. 1f, h-i. c Cell-cycle progression in samples presented in (a) and Fig. 1f, h-i. The percentage of cells with a 2n DNA content is indicated for each time point. (b and c) Data shown represent a single experiment. d Hi-C coverage (bin: 5 kb) in samples presented in (a) and Fig. 1f, h-i, as in Extended Data Fig. 1a. Vertical dotted line: DSB position. Horizontal dotted line: coverage of the undamaged control. e Heatmap of loop lengths and scores 4 hours post-DSB induction in control cells (untagged) or in Scc1-depleted cells simultaneously or 2 hours after DSB induction. Datasets subsampled at 15 million contacts each. f Contact probability as a function of genomic distance Pc(s) of cells 4 hours post-DSB induction in presence of Scc1 (that is untagged +DMSO or +IAA) or upon Scc1 depletion (that is AID tagged, +IAA added simultaneously or 2 hours after DSB induction). g Proportion of intra-chromosomal interactions for each 16 chromosomes from samples presented in (a) and Fig. 1f, h-i. In red: median ± interquartile range. * denotes statistical significance (p-value < 0.05) upon Mann-Whitney Wilcoxon rank sum test, two-tailed. h Western blot of Cdc45-(5xFlag)-AID probed with an anti-Flag antibody (top) and the loading control GAPDH (bottom) from samples presented in Fig. 1g-i. The arrow denotes Cdc45 and the * an aspecific cross-detection by the Flag antibody. i Cell-cycle progression determined by flow cytometry in samples presented in Fig. 1g-i. (h and i) Data shown represent 2 independent experiments. j Hi-C coverage binned at 5 kb of samples presented in Fig. 1g–i, as in Extended Data Fig. 1a. Data represent the mean of biological replicates. Vertical dotted line: position of the DSB. Horizontal dotted line: coverage of the undamaged control. k Aggregated ratio maps of 29 kb windows centered on centromeres over randomly chosen positions on the diagonal of the contact maps determined from data presented in Fig. 1g-i and from an untagged control. l Proportion of intra-chromosomal interactions for each 16 chromosomes from samples presented in Fig. 1g–i. In red: median. A damaged untagged control and an undamaged G1 control are shown for comparison. m Contact probability as a function of genomic distance Pc(s) in DMSO- or IAA-treated Cdc45-AID-tagged or untagged strains 4 hours post-DSB induction. n Heatmap of loop lengths and scores 4 hours post-DSB induction in Cdc45-AID tagged (+DMSO) and depleted (+IAA) cells, as well as in untagged cells shown for comparison. Loop lengths and scores decrease in the absence of the sister chromatid. Determined from datasets subsampled at 24 million contacts each.

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Extended Data Fig. 5 Determinants and organization of the LIP.

a Choreography of DNA repair proteins recruitment after DSB induction. b Hi-C coverage binned at 5 kb in a wild-type strain and resection mutants. Data are mean in wild-type (n=2), sgs1Δ (n=1), exo1Δ (n=1), exo1-D173A (n=1), and exo1Δ sgs1Δ (n=1). Vertical dotted line: position of the DSB. Horizontal dotted line: coverage of the undamaged control. c Contact maps (left, bin = 1 kb) the chr. V in a mec3Δ strain (APY569). d Hi-C coverage binned at 5 kb, as in Extended Data Fig. 1a., in nocodazole-treated wild-type and checkpoint deficient mutants (wild-type n=3, mre11Δ n=1, mec3Δ n=1, rad17Δ n=2, mec1Δ sml1Δ n=2 biological replicates). Other legends as b. e Contact maps (left, bin = 1 kb) and ratio map versus wild-type (bin = 2 kb) of a chr. V region in sml1Δ and mec1Δsml1Δ strains (APY737 and APY736, respectively). f Hi-C coverage binned at 5 kb, as in Extended Data Fig. 1a., in a wild-type strain and HR mutants (wild-type n=2, rad51Δ n=1, rad52Δ n=2 biological replicates). Other legends as b. g Contact maps (left, bin = 1 kb) and ratio map versus wild-type (bin = 2 kb) of a chr. V region in a rad52Δ strain (APY534). h Contact probability as a function of genomic distance Pc(s) in untreated wild-type, sgs1Δ, exo1Δ, exo1-D173A, exo1Δ sgs1Δ, rad51Δ, and rad52Δ strains (left), or in nocodazole-treated wild-type, mre11Δ, mec3Δ, rad17Δ, sml1Δ and mec1Δsml1Δ strains (right) from Extended Data Fig. 5 and Fig. 2.

Extended Data Fig. 6 Loop length increases in a pds5-AID mutant, highlighting strong cohesin roadblocks without affecting chromosome individualization.

a Contact maps (bin size: 1 kb) of a region of the undamaged Chr. XI in wild-type and hypomorphic pds5-AID strains 4 hours post-DSB induction on chr. V (APY466 and APY468, respectively). Generated from datasets containing ≃15-16 million genome-wide pairs each. Right panel: ratio map of the pds5-AID mutant over a wild-type strain. Dotted lines emanating from CEN11 highlight intra-chromosomal contacts made by the centromere. b Proportion of intra-chromosomal interactions for each 16 chromosomes in wild-type or pds5-AID strains 4 hours post-DSB induction. In red: median ± interquartile range. c Contact probability as a function of genomic distance (Pc(s), left) and its derivative (right). d Aggregated ratio maps of 29 kb windows centered on pairs of cohesin-enriched over randomly chosen positions in the same inter-distance range. Bin size: 1 kb. e Hi-C coverage binned at 5 kb as in Extended Data Fig. 1a. Data are mean for pds5-AID (n=2 biological replicates) and mean ±SEM for wild-type cells (n=3 biological replicates). Vertical dotted line: position of the DSB. Horizontal dotted line: coverage of the undamaged control. f Ratio map of a pds5-AID over a wild-type strain 4 hours post-DSB induction on chr. V (APY468 and APY466, respectively). Magnification of Fig. 3b. g Calibrated Scc1 ChIP-Seq profiles at the DSB region (left) or at an undamaged region on chr. V (right) in nocodazole-treated wild-type (APY875) and pds5-AID (APY878) strains 4 hours post-DSB induction. The Y-axis represents the mean number of calibrated reads per million within each 1 kb-bin. Data represents the mean ±SEM of biological replicates.

Extended Data Fig. 7 Cohesins are enriched at the DSB in a 9-1-1 clamp dependent manner.

a Top: Calibrated Scc1-Pk9 ChIP-Seq at the DSB region (left) or at an undamaged region on chr. V (right) in metaphase-arrested cells 4 hours following DSB induction or 4 hours following HO expression in a strain bearing an uncleavable HO cut-site (APY573 and APY667, respectively). Bottom: Difference of Scc1 enrichment in nocodazole-treated cells with and without a DSB (left axis) and resection profile computed from input coverage in nocodazole-treated cells 4 hours post-DSB induction (right axis). The 15-20 kb Scc1-enriched regions on each side of the DSB are highlighted in light-blue. Dotted black line: coverage of the undamaged input. Dotted blue line: median Scc1 ChIP difference ± DSB genome-wide. The DSB and centromere positions are indicated by red and white circles, respectively. b Contact maps (left, bin = 1 kb) the chr. V in a wild-type strain (APY332; 22.5 M contacts) and a Scc1-Pk9 tagged strain (APY573; 10 M contacts), 4 hours after DSB induction. c Contact probability as a function of genomic distance (Pc(s), left) and its derivative (right) of samples in (b). d Calibrated Scc1 ChIP-Seq profiles at the DSB region (left) or at an undamaged region on chr. V (right) in nocodazole-treated wild-type (APY573) and rad17Δ (APY648) strains 4 hours post-DSB induction. Other legends as in (a).

Extended Data Fig. 8 Study of homology search with Hi-C contact data.

a Hi-C coverage binned at 5 kb determined from samples presented in Fig. 4a and S8c. Data are mean ± SEM of biological replicates, except for the wild-type strain with a DSB on chr. IV (n=1). Vertical dotted line: position of the DSB. Horizontal dotted line: coverage of the undamaged control. b Contact probability as a function of genomic distance (Pc(s), left) and its derivative (right) of samples presented in Fig. 4a and S8c. c Same as in Fig. 4a, but with the DSB induced on chr. V. Representative of a biological replicate. Enriched long-range contacts detected between the DSB and the telomeres pointed by the black arrow. d Same as in Fig. 4c, but with the DSB induced on chr. V. Data represents the mean ±SEM of a biological replicate.

Extended Data Fig. 9 Study of homology search with Hi-C contact data.

a Rationale of the differential 4C-like ratio plots in Fig. 5. Example of the differential intra-chromosomal contacts made by the left DSB end 4 hours post-DSB induction (left; APY295) over undamaged metaphase-arrested cells (right; APY537), from matrices binned at 1 kb. ‘Stripes’ of 10 bins are selected in both matrices and each bin subsampled to match the lowest number of contacts of either matrix. The ratio between both stripes is performed by Serpentine12, which locally adjusts the binning to the coverage (see Methods). b 4C-like ratio plots of the left and right DSB end on chr IV and a control viewpoint (chr. XV) 4 hours post-DSB induction over undamaged metaphase-arrested cells (APY526 and APY537, respectively). The genome-wide (top) and intra-chromosomal (bottom) contact profiles are shown. Black arrow: DSB position. Black diamond: location of the control viewpoint. Representative biological replicate. c 4C-like ratio maps of contact frequencies of the left and right DSB regions 4 hours post-DSB induction in wild-type over rad51Δ cells (APY295 and APY525, respectively), genome-wide (top) or within chr. V (bottom). d Same as c. but comparing Scc1-depleted (Scc1-AID + IAA) vs. cells lacking a sister chromatid (Cdc45-AID +IAA, see Fig. 1g) (APY467 and APY513, respectively) 4 hours post-DSB induction. e Same as (c) but comparing rad17Δ over wild-type cells (APY780 and APY526 respectively) 4 hours post-DSB induction on chr. IV, upon nocodazole treatment. Representative biological replicate. f Same as (c) but comparing Scc1-depleted cells vs. nocodazole-treated rad17Δ cells (APY467 and APY542, respectively) 4 hours post-DSB induction on chr. V.

Extended Data Fig. 10 Cohesin promotes homology identification in cis and inhibits it in trans in multiple ways.

a Left: ratio map of wild-type over Scc1-depleted (Scc1-AID+IAA) cells 4 hours post-DSB induction of the left arm of chr V (APY466 and APY467, respectively). Right: ratio map of a pds5-AID mutant over a wt strain 4 hours post-DSB induction of the left arm of chr V (APY468 and APY466, respectively). Intersection of dotted lines corresponds to differential contacts between the DSB and the site used for introduction of the intra donor. No homology is present in the strains used to generate these maps. b Inhibition of D-loop formation at the inter-chromosomal donor upon presence of a 0.6 kb- and 2-kb long intra-chromosomal competitor. Data points show individual biological replicates. The mean is shown as a line. c Western blot of Scc1-(Pk3-)AID probed with an anti-Pk antibody (top) and a loading control GAPDH (bottom) over the course of IAA or DMSO (mock) treatment of samples presented in (d) and Fig. 6f. Data shown represent a single experiment. d Left: ratio of intra over inter D-loop levels in cells depleted (Scc1-AID + IAA) or not (wild-type + IAA) for cohesin (APY572 and APY570, respectively). Right: Fold change of intra and inter D-loop levels upon Scc1 depletion. The intra donor is 2 kb-long. Data points show individual biological replicates. Mean: line. e Same as in Fig. 6b but with an intra-chromosomal donor located on the other side of the DSB. The DSB-donor distance is equivalent to that with a donor at can1. f Left: ratio map of pds5-AID mutant vs. WT 4 hours post-DSB induction of the left arm of chr V (APY468 and APY466, respectively). Intersection of dotted lines corresponds to differential contacts between the DSB region and the site used for introduction of the intra donor located on the other side of the centromere. No homology is present in the strains used to generate these maps. Right: Hi-C contact frequency (bin = 10 kb) between the left DSB end and the can1 locus without a donor (left) in wild-type and pds5-AID cells at 4 hours post-DSB induction (APY295 and APY468, respectively). Each point represents a value (left) or a median of values (right) from independent Hi-C experiments (wild-type n=9 and pds5-AID n=2 biological replicates. In red: median and interquartile range. g Left: same as Fig. 6g but with the intra donor located on the right arm of chr. V, as shown in (e). Data represent n=4 and 2 biological replicates at 2 and 4 hours, respectively. Right: intra over inter donor preference as a function of Hi-C contact frequency. Similarly to Fig. 6h but the DLC data and contact data are at 4 hours. Data points show individual biological replicates.

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41556_2021_783_MOESM4_ESM.xlsx

Supplementary Table 1: Genotype of the Saccharomyces cerevisiae strains used in this study. Supplementary Table 2: Primers used in this study. Supplementary Table 3: Hi-C libraries generated in this study, and presentation in figures.

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Piazza, A., Bordelet, H., Dumont, A. et al. Cohesin regulates homology search during recombinational DNA repair. Nat Cell Biol 23, 1176–1186 (2021). https://doi.org/10.1038/s41556-021-00783-x

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