Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens
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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-07-01
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| Series: | Cancers |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2072-6694/14/15/3676 |
| _version_ | 1797442663564181504 |
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| author | Christopher A. Koczor Aaron J. Haider Kate M. Saville Jianfeng Li Joel F. Andrews Alison V. Beiser Robert W. Sobol |
| author_facet | Christopher A. Koczor Aaron J. Haider Kate M. Saville Jianfeng Li Joel F. Andrews Alison V. Beiser Robert W. Sobol |
| author_sort | Christopher A. Koczor |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T12:45:13Z |
| format | Article |
| id | doaj.art-c37aa81ac506447c91742c72ee98d4a9 |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-09T12:45:13Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-c37aa81ac506447c91742c72ee98d4a92023-11-30T22:13:56ZengMDPI AGCancers2072-66942022-07-011415367610.3390/cancers14153676Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound ScreensChristopher A. Koczor0Aaron J. Haider1Kate M. Saville2Jianfeng Li3Joel F. Andrews4Alison V. Beiser5Robert W. Sobol6Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USAMitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USADepartment of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USADepartment of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USAMitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USADepartment of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USADepartment of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USAPoly(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.https://www.mdpi.com/2072-6694/14/15/3676Poly(ADP-ribose)PARLivePARWWEsplit luciferaseBER |
| spellingShingle | Christopher A. Koczor Aaron J. Haider Kate M. Saville Jianfeng Li Joel F. Andrews Alison V. Beiser Robert W. Sobol Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens Cancers Poly(ADP-ribose) PAR LivePAR WWE split luciferase BER |
| title | Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens |
| title_full | Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens |
| title_fullStr | Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens |
| title_full_unstemmed | Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens |
| title_short | Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens |
| title_sort | live cell detection of poly adp ribose for use in genetic and genotoxic compound screens |
| topic | Poly(ADP-ribose) PAR LivePAR WWE split luciferase BER |
| url | https://www.mdpi.com/2072-6694/14/15/3676 |
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