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Using LacO Arrays to Monitor DNA Double-Strand Break Dynamics in Live Schizosaccharomyces pombe Cells

  • Bryan A. Leland
  • Megan C. King
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1176)

Abstract

LacO arrays, when combined with LacI-GFP, have been a valuable tool for studying nuclear architecture and chromatin dynamics. Here, we outline an experimental approach to employ the LacO/LacI-GFP system in S. pombe to assess DNA double-strand break (DSB) dynamics and the contribution of chromatin state to DSB repair. Previously, integration of long, highly repetitive LacO arrays in S. pombe has been a challenge. To address this problem, we have developed a novel approach, based on the principles used for homologous recombination-based genome engineering in higher eukaryotes, to integrate long, repetitive LacO arrays with targeting efficiencies as high as 70 %. Combining this facile LacO/LacI-GFP system with a site-specific, inducible DSB provides a means to monitor DSB dynamics at engineered sites within the genome.

Key words

LacO/LacI Chromatin dynamics DNA double-strand break Genome instability Homologous recombination Live-cell imaging Genome engineering S. pombe 

Notes

Acknowledgements

We would like to thank the Gasser lab and Russell labs for providing plasmids. This work was supported by the G. Harold and Leila Y. Mathers Charitable Foundation and the Searle Scholar Program (to M.C.K) and an NIGMS training grant T32GM007223 (to B.A.L.).

References

  1. 1.
    Negrini S, Gorgoulis VG, Halazonetis TD (2010) Genomic instability–an evolving hallmark of cancer. Nat Rev Mol Cell Biol 11:220–228PubMedCrossRefGoogle Scholar
  2. 2.
    Szostak JW, Orr-Weaver TL, Rothstein RJ et al (1983) The double-strand-break repair model for recombination. Cell 33:25–35PubMedCrossRefGoogle Scholar
  3. 3.
    Barzel A, Kupiec M (2008) Finding a match: how do homologous sequences get together for recombination? Nat Rev Genet 9:27–37PubMedCrossRefGoogle Scholar
  4. 4.
    Gehlen LR, Gasser SM, Dion V (2011) How broken DNA finds its template for repair: a computational approach. Prog Theor Phys Suppl 191:20–29CrossRefGoogle Scholar
  5. 5.
    Dion V, Gasser SM (2013) Chromatin movement in the maintenance of genome stability. Cell 152:1355–1364PubMedCrossRefGoogle Scholar
  6. 6.
    Haber JE (2012) Mating-type genes and MAT switching in Saccharomyces cerevisiae. Genetics 191:33–64PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Goodarzi AA, Noon AT, Deckbar D et al (2008) ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. Mol Cell 31:167–177PubMedCrossRefGoogle Scholar
  8. 8.
    Chiolo I, Minoda A, Colmenares SU et al (2011) Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144: 732–744PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    van Attikum H, Fritsch O, Hohn B et al (2004) Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119:777–788PubMedCrossRefGoogle Scholar
  10. 10.
    Costelloe T, Louge R, Tomimatsu N et al (2012) The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature 489:581–584PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Nagai S, Dubrana K, Tsai-Pflugfelder M et al (2008) Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322: 597–602Google Scholar
  12. 12.
    Chen X, Cui D, Papusha A et al (2012) The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends. Nature 489:576–580PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Grewal SIS, Jia S (2007) Heterochromatin revisited. Nat Rev Genet 8:35–46PubMedCrossRefGoogle Scholar
  14. 14.
    van der Oost J (2013) Molecular biology. New tool for genome surgery. Science 339:768–770Google Scholar
  15. 15.
    Rose MD, Winston FM, Heiter P (1990) Methods in yeast genetics: a laboratory course manual, Cold Spring Harbor Laboratory Protocols. Cold Spring Harbor, NYGoogle Scholar
  16. 16.
    Du L-L, Nakamura TM, Moser BA et al (2003) Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes. Mol Cell Biol 23:6150–6158PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194: 795–823PubMedCrossRefGoogle Scholar
  18. 18.
    Rohner S, Gasser SM, Meister P (2008) Modules for cloning-free chromatin tagging in Saccharomyces cerevisiae. Yeast 25:235–239PubMedCrossRefGoogle Scholar
  19. 19.
    Straight AF, Belmont AS, Robinett CC et al (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6:1599–1608PubMedCrossRefGoogle Scholar
  20. 20.
    Robinett CC, Straight A, Li G et al (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135:1685–1700PubMedCrossRefGoogle Scholar
  21. 21.
    Belmont AS (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11: 250–257PubMedCrossRefGoogle Scholar
  22. 22.
    Bähler J, Wu JQ, Longtine MS et al (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943–951PubMedCrossRefGoogle Scholar
  23. 23.
    Pâques F, Haber JE (1997) Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. Mol Cell Biol 17:6765–6771PubMedCentralPubMedGoogle Scholar
  24. 24.
    Basi G, Schmid E, Maundrell K (1993) TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123: 131–136PubMedCrossRefGoogle Scholar
  25. 25.
    Jovtchev G, Watanabe K, Pecinka A et al (2008) Size and number of tandem repeat arrays can determine somatic homologous pairing of transgene loci mediated by epigenetic modifications in Arabidopsis thaliana nuclei. Chromosoma 117:267–276PubMedCrossRefGoogle Scholar
  26. 26.
    Towbin BD, Meister P, Pike BL et al (2010) Repetitive transgenes in C. elegans accumulate heterochromatic marks and are sequestered at the nuclear envelope in a copy-number- and lamin-dependent manner. Cold Spring Harb Symp Quant Biol 75:555–565PubMedCrossRefGoogle Scholar
  27. 27.
    Nabeshima K, Kurooka H, Takeuchi M et al (1995) p93dis1, which is required for sister chromatid separation, is a novel microtubule and spindle pole body-associating protein phosphorylated at the Cdc2 target sites. Genes Dev 9:1572–1585PubMedCrossRefGoogle Scholar
  28. 28.
    Nabeshima K, Nakagawa T, Straight AF et al (1998) Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle. Mol Biol Cell 9: 3211–3225PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Hayles J, Nurse P (1992) Genetics of the fission yeast Schizosaccharomyces pombe. Annu Rev Genet 26:373–402PubMedCrossRefGoogle Scholar
  30. 30.
    Siam R, Dolan WP, Forsburg SL (2004) Choosing and using Schizosaccharomyces pombe plasmids. Methods 33:189–198Google Scholar
  31. 31.
    Sunder S, Greeson-Lott NT, Runge KW et al (2012) A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe. Yeast 29:275–291PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Watson AT, Werler P, Carr AM (2011) Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus. Gene 484:75–85PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Cell BiologyYale University School of MedicineNew HavenUSA

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