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Published on: June 9, 2017
Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens
Christopher A Koczor1,2, Aaron J Haider2, Kate M Saville1,2
1Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.
Abstract:
Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells. Here, we present two methods that expand on the use of LivePAR and its WWE domain. First, LivePAR enriches in the nucleus of cells following genotoxic challenge. Image quantitation can identify single-cell PAR formation following genotoxic stress at concentrations lower than PAR ELISA or PAR immunoblot, with greater sensitivity to genotoxic stress than CometChip. In a second approach, we used the RNF146-encoded WWE domain to develop a split luciferase probe for analysis in a 96-well plate assay. We then applied these PAR analysis tools to demonstrate their broad applicability. First, we show that both approaches can identify genetic modifications that alter PARylation levels, such as hyper-PARylation in BRCA2-deficient cancer cells. Second, we demonstrate the utility of the WWE split luciferase assay to characterize the cellular response of genotoxins, PARP inhibitors, and PARG inhibitors, thereby providing a screening method to identify PAR modulating compounds.
Insights
New tools quantify poly(ADP-ribose) (PAR) in live cells, aiding DNA repair studies. These methods offer sensitive detection of PAR formation and cellular responses to genotoxic stress and PAR-modulating compounds.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Poly(ADP-ribose) (PAR) is crucial for DNA repair complex formation.
- Sensitive quantification of PAR in live cells remains a challenge.
- Existing methods like ELISA and immunoblotting have limitations in sensitivity and live-cell analysis.
Purpose of the Study:
- To develop and validate novel tools for sensitive quantification of PAR in live cells.
- To expand the utility of the LivePAR probe and its WWE domain for PAR analysis.
- To assess cellular responses to genotoxic stress, PARP inhibitors, and PARG inhibitors.
Main Methods:
- Utilized the LivePAR probe (EGFP-WWE domain) for live-cell imaging of PAR formation at DNA damage sites.
- Developed a split luciferase probe based on the RNF146-encoded WWE domain for 96-well plate assays.
- Applied these tools to analyze PARylation levels in genetically modified cells and cellular responses to various compounds.
Main Results:
- LivePAR enables sensitive, single-cell detection of PAR formation following genotoxic stress, surpassing existing methods in sensitivity.
- The WWE split luciferase assay effectively identifies genetic alterations affecting PARylation, such as in BRCA2-deficient cells.
- Both methods demonstrate utility in characterizing cellular responses to genotoxins, PARP inhibitors, and PARG inhibitors.
Conclusions:
- The developed LivePAR and WWE split luciferase probes provide sensitive and versatile tools for quantifying PAR in live cells.
- These assays facilitate the study of DNA repair mechanisms and the screening of compounds that modulate PARylation.
- The tools offer improved sensitivity and applicability for research in cancer biology and drug discovery.
