PT  - JOURNAL ARTICLE
AU  - Thomas, Alix
AU  - Upadhyaya, Kapil
AU  - Bejan, Daniel
AU  - Adoff, Hayden
AU  - Cohen, Michael
AU  - Schultz, Carsten
TI  - A genetically encoded sensor for real-time monitoring of poly-ADP-ribosylation dynamics in-vitro and in cells
AID  - 10.1101/2024.06.11.598597
DP  - 2024 Jan 01
TA  - bioRxiv
PG  - 2024.06.11.598597
4099  - http://biorxiv.org/content/early/2024/06/13/2024.06.11.598597.short
4100  - http://biorxiv.org/content/early/2024/06/13/2024.06.11.598597.full
AB  - ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) groups to proteins, is a conserved post-translational modification (PTM) that occurs most prominently in response to DNA damage. ADP-ribosylation is a dynamic PTM regulated by writers (PARPs), erasers (ADPr hydrolases), and readers (ADPR binders). PARP1 is the primary DNA damage-response writer responsible for adding a polymer of ADPR to proteins (PARylation). Real-time monitoring of PARP1-mediated PARylation, especially in live cells, is critical for understanding the spatial and temporal regulation of this unique PTM. Here, we describe a genetically encoded FRET probe (pARS) for semi-quantitative monitoring of PARylation dynamics. pARS feature a PAR-binding WWE domain flanked with turquoise and Venus. With a ratiometric readout and excellent signal-to-noise characteristics, we show that pARS can monitor PARP1-dependent PARylation temporally and spatially in real-time. pARS provided unique insights into PARP1-mediated PARylation kinetics in vitro and high-sensitivity detection of PARylation in live cells, even under mild DNA damage. We also show that pARS can be used to determine the potency of PARP inhibitors in vitro and, for the first time, in live cells in response to DNA damage. The robustness and ease of use of pARS make it an important tool for the PARP field.Competing Interest StatementThe authors have declared no competing interest.