Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae

  • Sonia Silva
  • Irene Gallina
  • Nadine Eckert-Boulet
  • Michael LisbyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 920)


Fluorescence microscopy of the DNA damage response in living cells stands out from many other DNA repair assays by its ability to monitor the response to individual DNA lesions in single cells. This is particularly true in yeast, where the frequency of spontaneous DNA lesions is relatively low compared to organisms with much larger genomes such as mammalian cells. Single cell analysis of individual DNA lesions allows specific events in the DNA damage response to be correlated with cell morphology, cell cycle phase, and other specific characteristics of a particular cell. Moreover, fluorescence live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction of site-specific DNA lesions.

Key words

Homologous recombination Checkpoint Fluorescence microscopy 



We thank Dr. Neta Dean, Stony Brook University, for sharing the yEmRFP construct. This work was supported by Fundação para a Ciência e a Tecnologia (SS), The Danish Agency for Science, Technology and Innovation (ML, NEB), the Villum Kann Rasmussen Foundation (ML), and the European Research Council (ML).


  1. 1.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909PubMedCrossRefGoogle Scholar
  2. 2.
    Remington SJ (2006) Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16:714–721PubMedCrossRefGoogle Scholar
  3. 3.
    Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395PubMedCrossRefGoogle Scholar
  4. 4.
    Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182PubMedCrossRefGoogle Scholar
  5. 5.
    Keppler-Ross S, Noffz C, Dean N (2008) A new purple fluorescent color marker for genetic studies in Saccharomyces cerevisiae and Candida albicans. Genetics 179:705–710PubMedCrossRefGoogle Scholar
  6. 6.
    Lisby M, Rothstein R, Mortensen UH (2001) Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci U S A 98:8276–8282PubMedCrossRefGoogle Scholar
  7. 7.
    Alvaro D, Sunjevaric I, Reid RJ, Lisby M, Stillman DJ, Rothstein R (2006) Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries. Yeast 23:1097–1106PubMedCrossRefGoogle Scholar
  8. 8.
    Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, Jentsch S, Rothstein R, Aragon L, Lisby M (2007) The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol 9:923–931PubMedCrossRefGoogle Scholar
  9. 9.
    Andreson BL, Gupta A, Georgieva BP, Rothstein R (2010) The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response to DNA damage. Nucleic Acids Res 38:6490–6501PubMedCrossRefGoogle Scholar
  10. 10.
    Germann SM, Oestergaard VH, Haas C, Salis P, Motegi A, Lisby M (2011) Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination. DNA Repair (Amst) 10:210–224CrossRefGoogle Scholar
  11. 11.
    Reid R, Lisby M, Rothstein R (2002) Cloning-free genome alterations in Saccharomyce cerevisiae using adaptamer-mediated PCR. Methods Enzymol 350:258–277PubMedCrossRefGoogle Scholar
  12. 12.
    Zhao X, Muller EG, Rothstein R (1998) A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell 2:329–340PubMedCrossRefGoogle Scholar
  13. 13.
    Eckert-Boulet N, Rothstein R, Lisby M (2011) Cell biology of homologous recombination in yeast. Methods Mol Biol 745:523–536PubMedCrossRefGoogle Scholar
  14. 14.
    Gordon A, Colman-Lerner A, Chin TE, Benjamin KR, Yu RC, Brent R (2007) Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nat Methods 4:175–181PubMedCrossRefGoogle Scholar
  15. 15.
    Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572PubMedCrossRefGoogle Scholar
  16. 16.
    Lim CR, Kimata Y, Oka M, Nomaguchi K, Kohno K (1995) Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin NSP1. J Biochem (Tokyo) 118:13–17Google Scholar
  17. 17.
    Lisby M, Barlow JH, Burgess RC, Rothstein R (2004) Choreography of the DNA damage response; spatiotemporal relationships among checkpoint and repair proteins. Cell 118:699–713PubMedCrossRefGoogle Scholar
  18. 18.
    Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  19. 19.
    Reichard P (1988) Interactions between deoxyribonucleotide and DNA synthesis. Annu Rev Biochem 57:349–374PubMedCrossRefGoogle Scholar
  20. 20.
    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 412:557–561PubMedCrossRefGoogle Scholar
  21. 21.
    Lisby M, Mortensen UH, Rothstein R (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 5:572–577PubMedCrossRefGoogle Scholar
  22. 22.
    Beranek DT, Weis CC, Swenson DH (1980) A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis 1:595–606PubMedCrossRefGoogle Scholar
  23. 23.
    Friedland W, Jacob P, Paretzke HG, Merzagora M, Ottolenghi A (1999) Simulation of DNA fragment distributions after irradiation with photons. Radiat Environ Biophys 38:39–47PubMedCrossRefGoogle Scholar
  24. 24.
    Fronza G, Campomenosi P, Iannone R, Abbondandolo A (1992) The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions. Nucleic Acids Res 20:1283–1287PubMedCrossRefGoogle Scholar
  25. 25.
    Wiltrout ME, Walker GC (2011) The DNA polymerase activity of Saccharomyces cerevisiae Rev1 is biologically significant. Genetics 187:21–35PubMedCrossRefGoogle Scholar
  26. 26.
    Deng C, Brown JA, You D, Brown JM (2005) Multiple endonucleases function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae. Genetics 170:591–600PubMedCrossRefGoogle Scholar
  27. 27.
    Eng WK, Faucette L, Johnson RK, Sternglanz R (1988) Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol Pharmacol 34:755–760PubMedGoogle Scholar
  28. 28.
    Moore CW, McKoy J, Dardalhon M, Davermann D, Martinez M, Averbeck D (2000) DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae. Genetics 154:1085–1099PubMedGoogle Scholar
  29. 29.
    Nielsen I, Bentsen IB, Lisby M, Hansen S, Mundbjerg K, Andersen AH, Bjergbaek L (2009) A Flp-nick system to study repair of a single protein-bound nick in vivo. Nat Methods 6:753–757PubMedCrossRefGoogle Scholar
  30. 30.
    McConnell Smith A, Takeuchi R, Pellenz S, Davis L, Maizels N, Monnat RJ Jr, Stoddard BL (2009) Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc Natl Acad Sci U S A 106:5099–5104PubMedCrossRefGoogle Scholar
  31. 31.
    Jensen RE, Herskowitz I (1984) Directionality and regulation of cassette substitution in yeast. Cold Spring Harbor Symp Quant Biol 49:97–104PubMedCrossRefGoogle Scholar
  32. 32.
    Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Sonia Silva
    • 1
  • Irene Gallina
    • 1
  • Nadine Eckert-Boulet
    • 1
  • Michael Lisby
    • 1
    Email author
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations