Skip to main content
SpringerLink
Log in

Replication forks reverse at high frequency upon replication stress in Physarum polycephalum

  • Research Article
  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

The addition of hydroxyurea after the onset of S phase allows replication to start and permits the successive detecting of replication-dependent joint DNA molecules and chicken foot structures in the synchronous nuclei of Physarum polycephalum. We find evidence for a very high frequency of reversed replication forks upon replication stress. The formation of these reversed forks is dependent on the presence of joint DNA molecules, the impediment of the replication fork progression by hydroxyurea, and likely on the propensity of some replication origins to reinitiate replication to counteract the action of this compound. As hydroxyurea treatment enables us to successively detect the appearance of joint DNA molecules and then of reversed replication forks, we propose that chicken foot structures are formed both from the regression of hydroxyurea-frozen joint DNA molecules and from hydroxyurea-stalled replication forks. These experiments underscore the transient nature of replication fork regression, which becomes detectable due to the hydroxyurea-induced slowing down of replication fork progression.

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
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Alvino GM, Collingwood D, Murphy JM, Delrow J, Brewer BJ, Raghuraman MK (2007) Replication in hydroxyurea: it’s a matter of time. Mol Cell Biol 27:6396–6406

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Anglana M, Apiou F, Bensimon A, Debatisse M (2003) Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing. Cell 114:385–394

    Article  CAS  PubMed  Google Scholar 

  • Atkinson J, Mc Glynn P (2009) Replication fork reversal and the maintenance of genome stability. Nucleic Acids Res 37:3475–3492

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bénard M, Lagnel C, Pallotta D, Pierron G (1996) Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum. Mol Cell Biol 16:968–976

    PubMed Central  PubMed  Google Scholar 

  • Bénard M, Maric C, Pierron G (2001) DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum. Mol Cell 7:971–980

    Article  PubMed  Google Scholar 

  • Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 11:208–219

    Article  CAS  PubMed  Google Scholar 

  • Cotta-Ramusino C, Fachinetti D, Lucca C, Doksali Y, Lopes M, Sogo J, Foiani M (2005) Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells. Mol Cell 17:135–159

    Article  Google Scholar 

  • Courbet S, Gay S, Arnoult N, Wronka G, Anglana M, Brison O, Debatisse M (2008) Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature 455:557–560

    Article  CAS  PubMed  Google Scholar 

  • Cox MM, Goodman MF, Kreuser KN, Sheratt DJ, Sandler SJ, Marians KJ (2000) The importance of repairing stalled replication forks. Nature 404:37–41

    Article  CAS  PubMed  Google Scholar 

  • De Piccoli G, Katou Y, Itoh T, Nakato R, Shirahige K, Labib K (2012) Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases. Mol Cell 45:696–704

    Article  PubMed  Google Scholar 

  • Fachinetti D, Bermejo R, Cocito A, Minardi S, Katou Y, Kanoh Y, Shirahige K, Azvolinsky A, Zakian VA, Foiani M (2010) Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements. Mol Cell 39:595–605

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Feng W, Collingwood D, Boeck ME, Fox LA, Alvino G, Fangman WL, Raghuraman MK, Brewer BJ (2006) Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication. Nat Cell Biol 8:148–155

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fierro-Fernandez M, Hernandez P, Krimer DB, Schvartzman JB (2007a) Replication fork reversal occurs spontaneously after digestion but is constrained in supercoiled domains. J Biol Chem 282:18190–18196

    Article  CAS  PubMed  Google Scholar 

  • Fierro-Fernandez M, Hernandez P, Krimer DB, Stasiak A, Schvartzman JB (2007b) Topological locking restrains replication fork reversal. Proc Natl Acad Sci U S A 104:1500–1505

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Follonier C, Oehler J, Herrador R, Lopes M (2013) Friedreich’s ataxia-associated GAA repeats induce replication-fork reversal and unusual molecular junctions. Nat Struct Mol Biol 20:486–495

    Article  CAS  PubMed  Google Scholar 

  • Fouquet H, Bohme R, Wick R, Sauer HW (1975) Some evidence for replication-transcription coupling in Physarum polycephalum. J Cell Sci 18:27–39

    CAS  PubMed  Google Scholar 

  • Gilbert DM (2007) Replication origin plasticity, Taylor-made: inhibition vs recruitment of origins under conditions of replication stress. Chromosoma 116:341–347

    Article  PubMed  Google Scholar 

  • Hegnauer AM, Hustedt N, Shimada K, Pike BL, Vogel M, Amsler P, Rubin SM, Van Leeuwen F, Guénolé A, Van Attikum H, Thomä N, Gasser SM (2012) An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks. EMBO J 31:3768–3783

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hu J, Sun L, Shen F, Chen Y, Hua Y, Liu Y, Zhang M, Hu Y, Wang Q, Xu W, Sun F, Ji J, Murray JM, Carr AM, Kong D (2012) The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing. Cell 149:1221–1232

    Article  CAS  PubMed  Google Scholar 

  • Lai MS, Foiani M (2012) Dna2 offers support for stalled forks. Cell 149:1181–1183

    Article  CAS  PubMed  Google Scholar 

  • Liberi G, Cotta-Ramusino C, Lopes M, Sogo J, Conti C, Bensimon A, Foiani M (2006) Methods to study replication fork collapse in budding yeast. Methods Enzymol 409:442–462

    Article  CAS  PubMed  Google Scholar 

  • Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, Muzi-Falconi M, Newlon CS, Foiani M (2001) The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412:557–561

    Article  CAS  PubMed  Google Scholar 

  • Lopes M, Cotta-Ramusino C, Liberi G, Foiani M (2003) Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms. Mol Cell 12:1499–1510

    Article  CAS  PubMed  Google Scholar 

  • Maric C, Benard M, Pierron G (2010) RNase-dependent discontinuities associated with the crossovers of spontaneously formed joint DNA molecules in Physarum polycephalum. Chromosoma 119:601–611

    Article  CAS  PubMed  Google Scholar 

  • Mc Glynn P, Lloyd RG, Marians KJ (2001) Formation fo Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled. Proc Natl Acad Sci U S A 98:8235–8240

    Article  CAS  Google Scholar 

  • Neelsen KJ, Zanini IMY, Herrador R, Lopes M (2013a) Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates. J Cell Biol 2000:699–708

    Article  Google Scholar 

  • Neelsen KJ, Zanini IMY, Mijic S, Herrador R, Zellweger R, Ray Chaudhuri A, Creavin KD, Blow JJ, Lopes M (2013b) Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template. Genes Dev 27:2537–2542

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nitani N, Yadani C, Yabuuchi H, Masukata H, Nakagawa T (2008) Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks. Proc Natl Acad Sci U S A 105:12973–12978

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Postow L, Crisona NJ, Peter BJ, Hardy CD, Cozzarelli NR (2001) Topological challenges to DNA replication: conformations at the fork. Proc Natl Acad Sci U S A 98:8219–8226

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ray Chaudhuri A, Hashimoto Y, Herrador R, Neelsen KJ, Fachinetti D, Bermejo R, Cocito A, Costanzo V, Lopes M (2012) Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat Struct Mol Biol 19:417–423

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Vengrova S, Dalgaard JZ (2004) RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching. Genes Dev 18:794–804

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dana Branzei for constructive discussions of our data and Gérard Pierron for critical analyses of our results. We also thank Marie-Noëlle Prioleau and Dominique Weil for their great supports. We are very grateful to Terence Strick for critical reading of the manuscript and to the reviewers for helping us in improving our work. This work was supported by a general funding from the CNRS, grant L494 from Association de la Recherche Contre le Cancer, and grant ORC454 from Ligue Nationale Contre le Cancer.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Institut Jacques Monod, UMR 7592 CNRS-Université Paris Diderot-Paris7, 15 Rue Hélène Brion, 75205, Paris CEDEX 13, France

    Chrystelle Maric

  2. Laboratoire Biologie du Développement, UMR 7622 CNRS-UPMC-Paris6, 7 Quai Saint-Bernard, 75005, Paris, France

    Marianne Bénard

Authors
  1. Chrystelle Maric
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marianne Bénard
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chrystelle Maric.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 14.9 kb)

Fig. S1

(JPG 122 kb)

High resolution image (TIFF 125 kb)

Fig. S2

(JPG 175 kb)

High resolution image (TIFF 127 kb)

Fig. S3

(JPG 184 kb)

High resolution image (TIFF 167 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maric, C., Bénard, M. Replication forks reverse at high frequency upon replication stress in Physarum polycephalum . Chromosoma 123, 577–585 (2014). https://doi.org/10.1007/s00412-014-0471-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00412-014-0471-z

Keywords

Search

Navigation