Protocol

DNA Repair Protocols

Volume 920 of the series Methods in Molecular Biology pp 433-443

Date:

Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae

  • Sonia SilvaAffiliated withDepartment of Biology, University of Copenhagen
  • , Irene GallinaAffiliated withDepartment of Biology, University of Copenhagen
  • , Nadine Eckert-BouletAffiliated withDepartment of Biology, University of Copenhagen
  • , Michael LisbyAffiliated withDepartment of Biology, University of Copenhagen Email author 

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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