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Topoisomerase I poisoning results in PARP-mediated replication fork reversal

Nature Structural & Molecular Biology volume 19, pages 417–423 (2012)Cite this article

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Abstract

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress–inducing chemotherapeutics.

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Figure 1: CPT-treated S. cerevisiae cells have slower fork progression but do not require DSB repair to complete DNA replication.
Figure 2: Top1 poisoning globally slows down and frequently stalls replication-fork progression in human cells.
Figure 3: Slow but continuous replication upon mild Top1 poisoning is mostly uncoupled from DSB formation and repair in human cells.
Figure 4: Top1 inhibition induces PARP-mediated fork reversal.
Figure 5: PARP inactivation leads to DSB formation at minimal CPT doses.
Figure 6: Model for replication interference by Top1 poisons and their synergistic effects with PARP inhibitors.

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References

  1. Koster, D.A., Crut, A., Shuman, S., Bjornsti, M.A. & Dekker, N.H. Cellular strategies for regulating DNA supercoiling: a single-molecule perspective. Cell 142, 519–530 (2010).

    Article  CAS  Google Scholar 

  2. Pommier, Y. Topoisomerase I inhibitors: camptothecins and beyond. Nat. Rev. Cancer 6, 789–802 (2006).

    Article  CAS  Google Scholar 

  3. Hsiang, Y.H., Lihou, M.G. & Liu, L.F. Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res. 49, 5077–5082 (1989).

    CAS  PubMed  Google Scholar 

  4. Koster, D.A., Palle, K., Bot, E.S., Bjornsti, M.A. & Dekker, N.H. Antitumour drugs impede DNA uncoiling by topoisomerase I. Nature 448, 213–217 (2007).

    Article  CAS  Google Scholar 

  5. Bryant, H.E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).

    Article  CAS  Google Scholar 

  6. Rouleau, M., Patel, A., Hendzel, M.J., Kaufmann, S.H. & Poirier, G.G. PARP inhibition: PARP1 and beyond. Nat. Rev. Cancer 10, 293–301 (2010).

    Article  CAS  Google Scholar 

  7. Lord, C.J. & Ashworth, A. The DNA damage response and cancer therapy. Nature 481, 287–294 (2012).

    Article  CAS  Google Scholar 

  8. Kummar, S. et al. Phase I study of PARP inhibitor ABT-888 in combination with topotecan in adults with refractory solid tumors and lymphomas. Cancer Res. 71, 5626–5634 (2011).

    Article  CAS  Google Scholar 

  9. Tentori, L. et al. Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma. FASEB J. 20, 1709–1711 (2006).

    Article  CAS  Google Scholar 

  10. Curtin, N.J. PARP inhibitors for cancer therapy. Expert Rev. Mol. Med. 7, 1–20 (2005).

    Article  Google Scholar 

  11. Katou, Y. et al. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078–1083 (2003).

    Article  CAS  Google Scholar 

  12. Fachinetti, D. et al. Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements. Mol. Cell 39, 595–605 (2010).

    Article  CAS  Google Scholar 

  13. Greenfeder, S.A. & Newlon, C.S. Replication forks pause at yeast centromeres. Mol. Cell. Biol. 12, 4056–4066 (1992).

    Article  CAS  Google Scholar 

  14. Reid, R.J., Benedetti, P. & Bjornsti, M.A. Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs. Biochim. Biophys. Acta 1400, 289–300 (1998).

    Article  CAS  Google Scholar 

  15. Doksani, Y., Bermejo, R., Fiorani, S., Haber, J.E. & Foiani, M. Replicon dynamics, dormant origin firing, and terminal fork integrity after double-strand break formation. Cell 137, 247–258 (2009).

    Article  CAS  Google Scholar 

  16. Zierhut, C. & Diffley, J.F. Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J. 27, 1875–1885 (2008).

    Article  CAS  Google Scholar 

  17. Ge, X.Q. & Blow, J.J. Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories. J. Cell Biol. 191, 1285–1297 (2010).

    Article  CAS  Google Scholar 

  18. Teicher, B.A. Next generation topoisomerase I inhibitors: rationale and biomarker strategies. Biochem. Pharmacol. 75, 1262–1271 (2008).

    Article  CAS  Google Scholar 

  19. Sartori, A.A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).

    Article  CAS  Google Scholar 

  20. Hashimoto, Y., Chaudhuri, A.R., Lopes, M. & Costanzo, V. Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat. Struct. Mol. Biol. 17, 1305–1311 (2010).

    Article  CAS  Google Scholar 

  21. Lopes, M. Electron microscopy methods for studying in vivo DNA replication intermediates. Methods Mol. Biol. 521, 605–631 (2009).

    Article  CAS  Google Scholar 

  22. Atkinson, J. & McGlynn, P. Replication fork reversal and the maintenance of genome stability. Nucleic Acids Res. 37, 3475–3492 (2009).

    Article  CAS  Google Scholar 

  23. Postow, L. et al. Positive torsional strain causes the formation of a four-way junction at replication forks. J. Biol. Chem. 276, 2790–2796 (2001).

    Article  CAS  Google Scholar 

  24. Higgins, N.P., Kato, K. & Strauss, B. A model for replication repair in mammalian cells. J. Mol. Biol. 101, 417–425 (1976).

    Article  CAS  Google Scholar 

  25. Sogo, J.M., Lopes, M. & Foiani, M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599–602 (2002).

    Article  CAS  Google Scholar 

  26. Zhang, Y.W. et al. Poly(ADP-ribose) polymerase and XPF-ERCC1 participate in distinct pathways for the repair of topoisomerase I-induced DNA damage in mammalian cells. Nucleic Acids Res. 39, 3607–3620 (2011).

    Article  CAS  Google Scholar 

  27. Sugimura, K., Takebayashi, S., Taguchi, H., Takeda, S. & Okumura, K. PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA. J. Cell Biol. 183, 1203–1212 (2008).

    Article  CAS  Google Scholar 

  28. Wang, Z.Q. et al. Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease. Genes Dev. 9, 509–520 (1995).

    Article  CAS  Google Scholar 

  29. de Feraudy, S., Revet, I., Bezrookove, V., Feeney, L. & Cleaver, J.E. A minority of foci or pan-nuclear apoptotic staining of gammaH2AX in the S phase after UV damage contain DNA double-strand breaks. Proc. Natl. Acad. Sci. USA 107, 6870–6875 (2010).

    Article  CAS  Google Scholar 

  30. Petermann, E. et al. Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase. Mol. Cell. Biol. 26, 3319–3326 (2006).

    Article  CAS  Google Scholar 

  31. Bermejo, R. et al. The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores. Cell 146, 233–246 (2011).

    Article  CAS  Google Scholar 

  32. Durkacz, B.W., Shall, S. & Irwin, J. The effect of inhibition of (ADP-ribose)n biosynthesis on DNA repair assayed by the nucleoid technique. Eur. J. Biochem. 121, 65–69 (1981).

    Article  CAS  Google Scholar 

  33. Hassa, P.O. & Hottiger, M.O. The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front. Biosci. 13, 3046–3082 (2008).

    Article  CAS  Google Scholar 

  34. Räschle, M. et al. Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134, 969–980 (2008).

    Article  Google Scholar 

  35. O'Connell, B.C. et al. A genome-wide camptothecin sensitivity screen identifies a mammalian MMS22L-NFKBIL2 complex required for genomic stability. Mol. Cell 40, 645–657 (2010).

    Article  CAS  Google Scholar 

  36. Bermejo, R. et al. Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev. 21, 1921–1936 (2007).

    Article  CAS  Google Scholar 

  37. Bermejo, R. et al. Genome-organizing factors Top2 and Hmo1 prevent chromosome fragility at sites of S phase transcription. Cell 138, 870–884 (2009).

    Article  CAS  Google Scholar 

  38. Pellicioli, A. et al. Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase. EMBO J. 18, 6561–6572 (1999).

    Article  CAS  Google Scholar 

  39. Stojic, L. et al. Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase. Genes Dev. 18, 1331–1344 (2004).

    Article  CAS  Google Scholar 

  40. Jackson, D.A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295 (1998).

    Article  CAS  Google Scholar 

  41. Hanada, K. et al. The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat. Struct. Mol. Biol. 14, 1096–1104 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

M.L. and A.R.C. are supported by a Swiss National Science Foundation grant (PP00A-114922). A.R.C. was supported by a European Molecular Biology Organization (EMBO) short-term fellowship. V.C. and Y.H. are supported by Cancer Research UK, the Lister Institute of Preventive Medicine, a European Research Council (ERC-206281) startup grant and the EMBO Young Investigator Program (YIP). We thank K. Shirahige and M. Shimamura for assistance with ChIP-chip map construction. We thank K. Meyer, M. Hottiger, C. Blenn, F. Althaus, P. Janscak and A. Sartori (University of Zurich) and J. Rouse (University of Dundee), R. Kanaar (Erasmus MC) and K. Caldecott (University of Sussex) for sharing reagents; N. Ray Chaudhuri for technical assistance; the Center for Microscopy and Image Analysis of the University of Zurich for technical assistance with EM; and all members of the Lopes lab for helpful discussions.

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Author notes

  1. Daniele Fachinetti & Rodrigo Bermejo

    Present address: Present addresses: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California, USA (D.F.); Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas, Universidad de Salamanca, Spain (R.B.).,

  2. Yoshitami Hashimoto and Raquel Herrador: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland

    Arnab Ray Chaudhuri, Raquel Herrador, Kai J Neelsen & Massimo Lopes

  2. London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UK

    Yoshitami Hashimoto & Vincenzo Costanzo

  3. IFOM, Istituto FIRC di Oncologia Molecolare (IFOM-IEO Campus), Milan, Italy

    Daniele Fachinetti, Rodrigo Bermejo & Andrea Cocito

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Contributions

A.R.C. conducted all experiments in yeast, human and mouse cells by fluorescence-activated cell sorting, western blot, two-dimensional gels, DNA fiber spreading, PFGE, and immunofluorescence and carried out the EM analysis in all systems. Y.H. conducted all experiments in X. laevis extracts, including DNA preps for EM analysis. R.H. contributed to all synchronization experiments in human cells, western blots and immunofluorescence stainings. K.J.N. established and optimized, in the Lopes lab, most approaches required for this project. D.F. and R.B. assisted A.R.C. in the design, execution and interpretation of ChIP-chip experiments. A.C. carried out the ChIP-chip bioinformatic analysis. V.C. supervised the X. laevis experiments and assisted M.L. in finalizing the manuscript. M.L. planned and supervised the project and wrote the paper.

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Correspondence to Massimo Lopes.

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Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods (PDF 8586 kb)

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Ray Chaudhuri, A., Hashimoto, Y., Herrador, R. et al. Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat Struct Mol Biol 19, 417–423 (2012). https://doi.org/10.1038/nsmb.2258

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