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Monitoring Poly(ADP-Ribosyl)ation in Response to DNA Damage in Live Cells Using Fluorescently Tagged Macrodomains

  • Rebecca Smith
  • Gyula Timinszky
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)

Abstract

Poly(ADP-ribosyl)ation (PARylation) is a dynamic posttranslational modification that is added and removed rapidly at sites of DNA damage. PARylation is important for numerous aspects of DNA repair including chromatin decondensation and protein recruitment. Visualization of PARylation levels after DNA damage induction is generally obtained using traditional immunofluorescent techniques on fixed cells, which results in limited temporal resolution. Here, we describe a microscopy-based method to track ADP-ribosylation at break sites. This method relies on DNA damage induction using a 405 nm FRAP laser on Hoechst-treated cells expressing GFP-tagged PAR-binding proteins, such as macrodomains where the recruitment of the PAR-binder to sites of DNA damage gives an indication of PARylation levels.

Key words

DNA damage response Poly(ADP-ribosyl)ation PARP1, macrodomain Live-cell imaging Microirradiation 

Notes

Acknowledgments

This work was supported by the Worldwide Cancer Research grant (#14-1315) and the Deutsche Forschungsgemeinschaft grant (TI 817/2-1) to G.T. The authors declare no competing financial interests.

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Physiological Chemistry, Biomedical Center MunichLudwig-Maximilians-Universität MünchenPlanegg-MartinsriedGermany
  2. 2.Institute of GeneticsBiological Research Centre of the Hungarian Academy of SciencesSzegedHungary

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