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A Flp-nick system to study repair of a single protein-bound nick in vivo

Nature Methods volume 6, pages 753–757 (2009)Cite this article

Abstract

We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I–DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I–DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.

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Figure 1: FlpH305L was recruited to and cleaved at the ectopic FRT site.
Figure 2: S phase–specific histone H2A phosphorylation at the FRT site and co-localization of Rad52 foci with the FRT site in S/G2 cells.

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Acknowledgements

We thank M. Gartenberg (Robert Wood Johnson Medical School) for plasmids pFV17D-H305L and pFV17D and yeast strain ELT3; and M. Schmid (University of Aarhus) for comments on the manuscript. This work was supported by grants from the Danish Research Council (FNU 21-04-0354 and FNU 272-07-0366), Novo Nordisk Foundation, Aase and Einar Danielsens Foundation and Augustinus Foundation to L.B.; I.N. is supported by The Danish Cancer Society (DS07014); I.B.B. is supported by the Lundbeck Foundation (53/06) and M.L. is supported by The Danish Agency for Science, Technology and Innovation and the Villum Kann Rasmussen Foundation.

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Authors and Affiliations

  1. Department of Molecular Biology, University of Aarhus, Aarhus, Denmark

    Ida Nielsen, Iben Bach Bentsen, Sabine Hansen, Kamilla Mundbjerg, Anni H Andersen & Lotte Bjergbaek

  2. Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Michael Lisby

Authors
  1. Ida Nielsen
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  2. Iben Bach Bentsen
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  3. Michael Lisby
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  4. Sabine Hansen
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  7. Lotte Bjergbaek
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Contributions

I.N., I.B.B., M.L., S.H., K.M. and L.B. carried out experiments. L.B. and A.H.A. designed the system. L.B. wrote the manuscript.

Corresponding author

Correspondence to Lotte Bjergbaek.

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Supplementary Figures 1–5, Supplementary Tables 1–2 and Supplementary Protocol (PDF 642 kb)

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Nielsen, I., Bentsen, I., Lisby, M. et al. A Flp-nick system to study repair of a single protein-bound nick in vivo. Nat Methods 6, 753–757 (2009). https://doi.org/10.1038/nmeth.1372

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