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A genetically encoded sensor for real-time monitoring of poly-ADP-ribosylation dynamics in-vitro and in cells

bioRxiv, ISSN: 2692-8205
2024
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A genetically encoded sensor for real-time monitoring of poly-ADP-ribosylation dynamics in-vitro and in cells

2024 JUN 28 (NewsRx) -- By a News Reporter-Staff News Editor at NewsRx Life Science Daily -- According to news reporting based on a preprint

Article Description

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.

Bibliographic Details

Thomas, Alix; Upadhyaya, Kapil; Bejan, Daniel; Adoff, Hayden; Cohen, Michael S; Schultz, Carsten

Cold Spring Harbor Laboratory

Biochemistry, Genetics and Molecular Biology; Agricultural and Biological Sciences; Immunology and Microbiology; Neuroscience; Pharmacology, Toxicology and Pharmaceutics

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