Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae

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

Abstract

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 

Notes

Acknowledgment

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).

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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
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagenDenmark

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