A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

  • Nicolai B. Larsen
  • Ian D. Hickson
  • Hocine W. MankouriEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1672)


Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an “alien” impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

Key words

Replication fork barrier DNA replication stress Tus-Ter 



Work in the authors’ laboratory is funded by the Danish National Research Foundation (DNRF115), The European Research Council, The Novo Nordisk Foundation, and The Nordea Foundation.


  1. 1.
    Hill TM, Marians KJ (1990) Escherichia coli Tus protein acts to arrest the progression of DNA replication forks in vitro. Proc Natl Acad Sci U S A 87(7):2481–2485CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Larsen NB, Hickson ID, Mankouri HW (2014) Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes. Cell Cycle 13(19):2994–2998. doi: 10.4161/15384101.2014.958912 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Larsen NB, Sass E, Suski C et al (2014) The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast. Nat Commun 5:3574. doi: 10.1038/ncomms4574 CrossRefPubMedGoogle Scholar
  4. 4.
    Willis NA, Chandramouly G, Huang B et al (2014) BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510(7506):556–559. doi: 10.1038/nature13295 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Willis NA, Scully R (2016) Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier. Cell Cycle:1–9. doi: 10.1080/15384101.2016.1172149
  6. 6.
    Natsume T, Kiyomitsu T, Saga Y et al (2016) Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep 15(1):210–218. doi: 10.1016/j.celrep.2016.03.001 CrossRefPubMedGoogle Scholar
  7. 7.
    Calzada A, Hodgson B, Kanemaki M et al (2005) Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev 19(16):1905–1919. doi: 10.1101/gad.337205 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ahn JS, Osman F, Whitby MC (2005) Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast. EMBO J 24(11):2011–2023. doi: 10.1038/sj.emboj.7600670 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lambert S, Watson A, Sheedy DM et al (2005) Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier. Cell 121(5):689–702. doi: 10.1016/j.cell.2005.03.022 CrossRefPubMedGoogle Scholar
  10. 10.
    Iraqui I, Chekkal Y, Jmari N et al (2012) Recovery of arrested replication forks by homologous recombination is error-prone. PLoS Genet 8(10):e1002976. doi: 10.1371/journal.pgen.1002976 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Liberi G, Cotta-Ramusino C, Lopes M et al (2006) Methods to study replication fork collapse in budding yeast. Methods Enzymol 409:442–462CrossRefPubMedGoogle Scholar
  12. 12.
    Mankouri HW, Huttner D, Hickson ID (2013) How unfinished business from S-phase affects mitosis and beyond. EMBO J 32(20):2661–2671. doi: 10.1038/emboj.2013.211 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Raghuraman MK, Winzeler EA, Collingwood D et al (2001) Replication dynamics of the yeast genome. Science 294(5540):115–121. doi: 10.1126/science.294.5540.115 CrossRefPubMedGoogle Scholar
  14. 14.
    Muller CA, Hawkins M, Retkute R et al (2014) The dynamics of genome replication using deep sequencing. Nucleic Acids Res 42(1):e3. doi: 10.1093/nar/gkt878 CrossRefPubMedGoogle Scholar
  15. 15.
    Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):31–34. doi: 10.1038/nprot.2007.13 CrossRefPubMedGoogle Scholar
  16. 16.
    Looke M, Kristjuhan K, Kristjuhan A (2011) Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 50(5):325–328. doi: 10.2144/000113672 PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Nicolai B. Larsen
    • 1
  • Ian D. Hickson
    • 1
  • Hocine W. Mankouri
    • 1
    Email author
  1. 1.The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

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