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

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.