Cell Biology of Homologous Recombination in Yeast

  • Nadine Eckert-Boulet
  • Rodney Rothstein
  • Michael Lisby
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

Abstract

Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces cerevisiae using fluorescence microscopy.

Key words

Homologous recombination fluorescence microscopy DNA damage DNA double-strand break repair 

Notes

Acknowledgments

The LacI-R197K mutant protein was engineered by Christian Müller. This work was supported by The Danish Agency for Science, Technology and Innovation (ML), the Villum Kann Rasmussen Foundation (ML), GM67055 (RR), and the Lundbeck Foundation (NEB).

References

  1. 1.
    Krogh, B. and Symington, L. (2004) Recombination proteins in yeast. Annu Rev Genet 38, 233–271.PubMedCrossRefGoogle Scholar
  2. 2.
    Lisby, M. and Rothstein, R. (2004) DNA damage checkpoint and repair centers. Curr Opin Cell Biol 16, 328–334.PubMedCrossRefGoogle Scholar
  3. 3.
    Lisby, M. and Rothstein, R. (2005) Localization of checkpoint and repair proteins in eukaryotes. Biochimie 87, 579–589.PubMedCrossRefGoogle Scholar
  4. 4.
    Lisby, M. and Rothstein, R. (2009) Choreography of recombination proteins during the DNA damage response. DNA Repair (Amst) 8, 1068–1076.CrossRefGoogle Scholar
  5. 5.
    Sherman, F. Fink, G.R., and Hicks, J.B. (1986) Methods in yeast genetics (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).Google Scholar
  6. 6.
    Moore, C.W., McKoy, J., Dardalhon, M., Davermann, D., Martinez, M., and Averbeck, D. (2000) DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae. Genetics 154, 1085–1099.PubMedGoogle Scholar
  7. 7.
    Thomas, B.J. and Rothstein, R. (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56, 619–630.PubMedCrossRefGoogle Scholar
  8. 8.
    Zhao, X., Muller, E.G., and Rothstein, R. (1998) A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell 2, 329–340.PubMedCrossRefGoogle Scholar
  9. 9.
    Erdeniz, N., Mortensen, U.H., and Rothstein, R. (1997) Cloning-free PCR-based allele replacement methods. Genome Res 7, 1174–1183.PubMedGoogle Scholar
  10. 10.
    Torres-Rosell, J., Sunjevaric, I., De Piccoli, G., Sacher, M., Eckert-Boulet, N., Reid, R., Jentsch, S., Rothstein, R., Aragon, L., and Lisby, M. (2007) The Smc5–Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol 9, 923–931.PubMedCrossRefGoogle Scholar
  11. 11.
    Reid, R., Lisby, M., and Rothstein, R. (2002) Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR. Methods Enzymol 350, 258–277.PubMedCrossRefGoogle Scholar
  12. 12.
    Lisby, M., Mortensen, U.H., and Rothstein, R. (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 5, 572–577.PubMedCrossRefGoogle Scholar
  13. 13.
    Jensen, R.E. and Herskowitz, I. (1984) Directionality and regulation of cassette substitution in yeast. Cold Spring Harb Symp Quant Biol 49, 97–104.PubMedGoogle Scholar
  14. 14.
    Hoffman, C.S. and Winston, F. (1987) A ten-minute DNA preparation efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272.PubMedCrossRefGoogle Scholar
  15. 15.
    Gietz, D., St Jean, A., Woods, R.A., and Schiestl, R.H. (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20, 1425.PubMedCrossRefGoogle Scholar
  16. 16.
    Straight, A.F., Belmont, A.S., Robinett, C.C., and Murray, A.W. (1996) GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion. Curr Biol 6, 1599–1608.PubMedCrossRefGoogle Scholar
  17. 17.
    Michaelis, C., Ciosk, R., and Nasmyth, K. (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35–45.PubMedCrossRefGoogle Scholar
  18. 18.
    Lisby, M., Rothstein, R., and Mortensen, U.H. (2001) Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci USA 98, 8276–8282.PubMedCrossRefGoogle Scholar
  19. 19.
    Lim, C.R., Kimata, Y., Oka, M., Nomaguchi, K., and 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, 13–17.Google Scholar
  20. 20.
    Lisby, M., Barlow, J.H., Burgess, R.C., and Rothstein, R. (2004) Choreography of the DNA damage response; spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713.PubMedCrossRefGoogle Scholar
  21. 21.
    Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., and Remington, S.J. (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395.PubMedCrossRefGoogle Scholar
  22. 22.
    Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., and Tsien, R.Y. (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99, 7877–7882.PubMedCrossRefGoogle Scholar
  23. 23.
    Heim, R. and Tsien, R.Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6, 178–182.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Nadine Eckert-Boulet
    • 1
  • Rodney Rothstein
    • 2
  • Michael Lisby
    • 1
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of Genetics and DevelopmentColumbia University Medical CenterNew YorkUSA

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