Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations

Nature Cell Biology volume 20, pages 92–103 (2018)Cite this article

Subjects

Abstract

The resolution of joint molecules that link recombining sister chromatids is essential for chromosome segregation. Here, we determine the fate of unresolved recombination intermediates arising in cells lacking two nucleases required for resolution (GEN1 –/– knockout cells depleted of MUS81). We find that intermediates persist until mitosis and form a distinct class of anaphase bridges, which we term homologous recombination ultra-fine bridges (HR-UFBs). HR-UFBs are distinct from replication stress-associated UFBs, which arise at common fragile sites, and from centromeric UFBs. HR-UFBs are processed by BLM helicase to generate single-stranded RPA-coated bridges that are broken during mitosis. In the next cell cycle, DNA breaks activate the DNA damage checkpoint response, and chromosome fusions arise by non-homologous end joining. Consequently, the cells undergo cell cycle delay and massive cell death. These results lead us to present a model detailing how unresolved recombination intermediates can promote DNA damage and chromosomal instability.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Phenotypic analysis of resolvase-deficient cells.
Fig. 2: Persistent recombination intermediates lead to the formation of HR-UFBs.
Fig. 3: Generation of UFBs by homologous recombination in resolvase-deficient cells.
Fig. 4: HR-UFB breakage leads to DNA damage and gross chromosome abnormalities.
Fig. 5: BLM is required for the formation of RPA-coated UFBs.
Fig. 6: Unwinding of UFBs by PICH and BLM facilitates cell division.
Fig. 7: General mechanism for the formation of single-stranded UFBs.

Similar content being viewed by others

References

  1. Uhlmann, F. SMC complexes: from DNA to chromosomes. Nat. Rev. Mol. Cell Biol. 17, 399–412 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Chan, K. L. & Hickson, I. D. New insights into the formation and resolution of ultra-fine anaphase bridges. Sem. Cell Dev. Biol. 22, 906–912 (2011).

    Article  CAS  Google Scholar 

  3. Baumann, C., Korner, R., Hofmann, K. & Nigg, E. A. PICH, a centromere-associated SNF2 family ATPase, is regulated by PLK1 and required for the spindle checkpoint. Cell 128, 101–114 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Chan, K. L., North, P. S. & Hickson, I. D. BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges. EMBO J. 26, 3397–3409 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chan, K. L. & Hickson, I. D. On the origins of ultra-fine anaphase bridges. Cell Cycle 8, 3065–3066 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Germann, S. M. et al. TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability. J. Cell Biol. 204, 45–59 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Liu, Y., Nielsen, C. F., Yao, Q. & Hickson, I. D. The origins and processing of ultra fine anaphase DNA bridges. Curr. Opin. Genet. Dev. 26, 1–5 (2014).

    Article  PubMed  Google Scholar 

  8. Nielsen, C. F. et al. PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis. Nat. Commun. 6, 8962 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wang, L. H., Schwarzbam, T., Speicher, M. R. & Nigg, E. A. Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation. Chromosoma 117, 123–135 (2008).

    Article  PubMed  Google Scholar 

  10. Chan, K. L., Palmai-Pallag, T., Ying, S. M. & Hickson, I. D. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat. Cell Biol. 11, 753–760 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Naim, V. & Rosselli, F. The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat. Cell Biol. 11, 761–768 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Naim, V., Wilhelm, T., Debatisse, M. & Rosselli, F. ERCC1 and MUS81-EME1 promote sister chromatid separation by processing late replication intermediates at common fragile sites during mitosis. Nat. Cell Biol. 15, 1008–1015 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Ying, S. M. et al. MUS81 promotes common fragile site expression. Nat. Cell Biol. 15, 1001–1007 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Barefield, C. & Karlseder, J. The BLM helicase contributes to telomere maintenance through processing of late-replicating intermediate structures. Nucl. Acids Res. 40, 7358–7367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Maciejowski, J., Li, Y., Bosco, N., Campbell, P. J. & de Lange, T. Chromothripsis and kataegis induced by telomere crisis. Cell 163, 1641–1654 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nera, B., Huang, H.-S., Lai, T. & Xu, L. X. Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions. Nat. Commun. 6, 10132 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sarbajna, S., Davies, D. & West, S. C. Roles of SLX1-SLX4, MUS81-EME1 and GEN1 in avoiding genome instability and mitotic catastrophe. Genes Dev. 28, 1124–1136 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wu, L. & Hickson, I. D. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Chen, X. B. et al. Human MUS81-associated endonuclease cleaves Holliday junctions in vitro. Mol. Cell 8, 1117–1127 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Ciccia, A., Constantinou, A. & West, S. C. Identification and characterization of the human MUS81/EME1 endonuclease. J. Biol. Chem. 278, 25172–25178 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Fekairi, S. et al. Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases. Cell 138, 78–89 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Munoz, I. M. et al. Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol. Cell 35, 116–127 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Svendsen, J. M. et al. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair. Cell 138, 63–77 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ip, S. C. Y. et al. Identification of Holliday junction resolvases from humans and yeast. Nature 456, 357–361 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Rass, U. et al. Mechanism of Holliday junction resolution by the human GEN1 protein. Genes Dev. 24, 1559–1569 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chan, Y. W. & West, S. C. GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism. Nucl. Acids Res. 43, 10882–10892 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wyatt, H. D. M., Laister, R. C., Martin, S. R., Arrowsmith, C. H. & West, S. C. The SMX DNA repair tri-nuclease. Mol. Cell 65, 848–860 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wyatt, H. D. M., Sarbajna, S., Matos, J. & West, S. C. Coordinated actions of SLX1-SLX4 and MUS81-EME1 for Holliday junction resolution in human cells. Mol. Cell 52, 234–247 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Wechsler, T., Newman, S. & West, S. C. Aberrant chromosome morphology in human cells defective for Holliday junction resolution. Nature 471, 642–646 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Castor, D. et al. Cooperative control of Holliday junction resolution and DNA repair by the SLX1 and MUS81-EME1 nucleases. Mol. Cell 52, 221–233 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Garner, E., Kim, Y., Lach, F. P., Kottemann, M. C. & Smogorzewska, A. Human GEN1 and the SLX4-associated nucleases MUS81 and SLX1 are essential for the resolution of replication-induced Holliday junctions. Cell Rep. 5, 207–215 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Duda, H. et al. A mechanism for controlled breakage of under-replicated chromosomes during mitosis. Dev. Cell 39, 740–755 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Matos, J., Blanco, M. G., Maslen, S. L., Skehel, J. M. & West, S. C. Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis. Cell 147, 158–172 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chan, Y. W. & West, S. C. Spatial control of the GEN1 Holliday junction resolvase ensures genome stability. Nat. Commun. 5, 5844 (2014).

    Article  Google Scholar 

  35. Nair, N., Castor, D., Macartney, T. & Rouse, J. Identification and characterization of MUS81 point mutations that abolish interaction with the SLX4 scaffold protein. DNA Rep. 24, 131–137 (2014).

    Article  CAS  Google Scholar 

  36. Minocherhomji, S. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Schlacher, K. et al. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145, 529–542 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lukas, C. et al. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat. Cell Biol. 13, 243–253 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Harrigan, J. A. et al. Replication stress induces 53BP1-containing OPT domains in G1 cells. J. Cell Biol. 193, 97–108 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Klein Douwel, D. et al. XPF-ERCC1 acts in unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4. Mol. Cell 54, 460–471 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Hodskinson, M. R. G. et al. Mouse SLX4 is a tumour suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair. Mol. Cell 54, 472–484 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Burrell, R. A. et al. Replication stress links structural and numerical cancer chromosomal instability. Nature 494, 492–496 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ke, Y. et al. PICH and BLM limit histone association with anaphase centromeric threads and promote their resolution. EMBO J. 30, 3309–3321 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hengeveld, R. C. et al. Rif1 Is required for resolution of ultrafine DNA bridges in anaphase to ensure genomic stability. Dev. Cell 34, 466–474 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Houchmandzadeh, B., Marko, J. F., Chatenay, D. & Libchaber, A. Elasticity and structure of eukaryote chromosomes studied by micromanipulation and micropipette aspiration. J. Cell Biol. 139, 1–12 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Alexander, S. P. & Rieder, C. L. Chromosome motion during attachment to the vertebrate spindle: initial saltatory-like behavior of chromosomes and quantitative analysis of force production by nascent kinetochore fibers. J. Cell Biol. 113, 805–815 (1991).

    Article  CAS  PubMed  Google Scholar 

  47. Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H. E. How strong is a covalent bond? Science 283, 1727–1730 (1999).

    Article  CAS  PubMed  Google Scholar 

  48. Bustamante, C., Smith, S. B., Liphardt, J. & Smith, D. R. Single-molecule studies of DNA molecules. Curr. Opin. Struct. Biol. 10, 279–285 (2000).

    Article  CAS  PubMed  Google Scholar 

  49. Mullins, J. M. & Biesele, J. J. Terminal phase of cytokinesis in D-98s cells. J. Cell Biol. 73, 672–684 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Steigemann, P. et al. Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 136, 473–484 (2009).

    Article  PubMed  Google Scholar 

  51. Janssen, A., van der Burg, M., Szuhai, K., Kops, G. J. & Medema, R. H. Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations. Science 333, 1895–1898 (2011).

    Article  CAS  PubMed  Google Scholar 

  52. Ly, P. et al. Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining. Nat. Cell Biol. 19, 68–75 (2017).

    Article  CAS  PubMed  Google Scholar 

  53. Thompson, S. L. & Compton, D. A. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell Biol. 180, 665–672 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Tanno, Y. et al. The inner centromere-shugoshin network prevents chromosomal instability. Science 349, 1237–1240 (2015).

    Article  CAS  PubMed  Google Scholar 

  55. Raderschall, E. et al. Formation of higher-order nuclear RAD51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis. J. Cell Sci. 115, 153–164 (2002).

    CAS  PubMed  Google Scholar 

  56. Klein, H. L. The consequences of RAD51 overexpression for normal and tumor cells. DNA Repair 7, 686–693 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Marsden, C. G. et al. The tumor-associated variant RAD51 G151D induces a hyper-recombination phenotype. PLoS Genet. 12, e1006208 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Rodriguez-Lopez, A. M., Whitby, M. C., Borer, C. M., Bachler, M. A. & Cox, L. S. Correction of proliferation and drug sensitivity defects in the progeriod Werner’s Syndrome by Holliday junction resolution. Rejuvenation Res. 10, 27–40 (2007).

    Article  CAS  PubMed  Google Scholar 

  59. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Essletzbichler, P. et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 24, 2059–2065 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Paliwal, S., Kanagaraj, R., Sturzenegger, A., Burdova, K. & Janscak, P. Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing. Nucl. Acids Res. 42, 2380–2390 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Chowdhury, D. et al. The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death. Mol. Cell 23, 133–142 (2006).

    Article  CAS  PubMed  Google Scholar 

  64. Fugger, K. et al. Human FBH1 helicase contributes to genome maintenance via pro- and anti-recombinase activities. J. Cell Biol. 186, 655–663 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Petermann, E., Orta, M. L., Issaeva, N., Schultz, N. & Helleday, T. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol. Cell 37, 492–502 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank members of the West Lab for their help and encouragement, and K. L. Chan (University of Sussex, UK) for sharing unpublished data. This work was supported by the Francis Crick Institute (FC10212), the European Research Council (ERC-ADG-249145 and ERC-ADG-666400) and the Louis-Jeantet Foundation. The Francis Crick Institute receives core funding from Cancer Research UK, the Medical Research Council and the Wellcome Trust. K.F. is the recipient of a fellowship from the Lundbeck Foundation.

Author information

Authors and Affiliations

  1. The Francis Crick Institute, London, UK

    Ying Wai Chan, Kasper Fugger & Stephen C. West

Authors
  1. Ying Wai Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kasper Fugger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephen C. West
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.W.C. carried out the experiments and was assisted by K.F with the fibre assays and some of the immunofluorescence experiments. Y.W.C. and S.C.W. designed the project and wrote the manuscript.

Corresponding author

Correspondence to Stephen C. West.

Ethics declarations

Competing interests

The authors have no competing financial interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Table 1 Legend.

Life Sciences Reporting Summary

Supplementary Table 1

Statistics Source Data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chan, Y.W., Fugger, K. & West, S.C. Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations. Nat Cell Biol 20, 92–103 (2018). https://doi.org/10.1038/s41556-017-0011-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-017-0011-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing