Related Experiment Video
Updated: May 6, 2026
09:23
Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast
Published on: November 28, 2018
6.5K
Visualization and Quantitative Analysis of Genotoxin-induced PARP1/PARP2 Activation in Cells using a Fluorescent
Wynand P Roos1, Rasha Q Al-Rahahleh1, Christopher A Koczor2
1Department of Pathology and Laboratory Medicine, Warren Alpert Medical School & Legorreta Cancer Center, Brown University.
Journal of Visualized Experiments : Jove
|May 4, 2026
Summary
Poly(ADP-ribose) (PAR) synthesis by PARP1/PARP2 is crucial for DNA repair. A new assay using PAR-binding domains (PBDs) fused to EGFP allows visualization and quantification of PAR accumulation during DNA damage response.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Poly(ADP-ribose) (PAR) is a polymer synthesized by PARP enzymes, primarily PARP1 and PARP2, in response to DNA breaks.
- PAR acts as a post-translational modification and can also modify DNA and RNA, playing a critical role in DNA repair and the DNA damage response (DDR).
- The dynamic regulation of PAR levels by synthesis (PARP) and degradation (PARG) is essential for cellular processes like DNA repair, replication, transcription, and cell death.
Purpose of the Study:
- To develop and describe an optimized assay for visualizing and quantifying PAR accumulation at sites of DNA damage.
- To utilize PAR-binding domains (PBDs) fused to enhanced green fluorescent protein (EGFP) for sensitive detection of PAR foci.
- To facilitate cellular analysis of PAR dynamics in response to genomic insults.
Main Methods:
- Development of a cell-based assay using a fragment of RNF146 containing the PBD/WWE domain fused to EGFP.
- Production of lentiviral particles for transduction of target mammalian cell lines.
- Induction of genomic DNA damage in treated cells, followed by confocal fluorescence microscopy and semi-automated data quantification.
Main Results:
- Successful visualization and quantification of PAR accumulation at sites of genomic insult and ongoing base excision repair (BER) or single-strand break repair (SSBR).
- Demonstration of the utility of EGFP-fused PBDs for optimizing cell-based quantitation of PAR foci.
- Establishment of a robust experimental workflow from lentiviral production to data analysis.
Conclusions:
- The developed assay provides a sensitive and quantitative method for studying PAR dynamics in cellular DNA damage response.
- EGFP-tagged PBDs are effective tools for visualizing PAR accumulation, aiding in the understanding of DNA repair mechanisms.
- This methodology can be applied to further investigate the roles of PAR in various DNA metabolic processes and cellular signaling pathways.
