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

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ADP-ribosylation and NAD+ Utilizing Enzymes

Part of the book series: Methods in Molecular Biology ((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.

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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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Authors and Affiliations

  1. Department of Physiological Chemistry, Biomedical Center Munich, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

    Rebecca Smith & Gyula Timinszky

  2. Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary

    Gyula Timinszky

Authors
  1. Rebecca Smith
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  2. Gyula Timinszky
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Corresponding author

Correspondence to Gyula Timinszky .

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Editors and Affiliations

  1. Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Paul Chang

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Smith, R., Timinszky, G. (2018). Monitoring Poly(ADP-Ribosyl)ation in Response to DNA Damage in Live Cells Using Fluorescently Tagged Macrodomains. In: Chang, P. (eds) ADP-ribosylation and NAD+ Utilizing Enzymes. Methods in Molecular Biology, vol 1813. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8588-3_2

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  • DOI: https://doi.org/10.1007/978-1-4939-8588-3_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8587-6

  • Online ISBN: 978-1-4939-8588-3

  • eBook Packages: Springer Protocols

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