51. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells
- Author
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Kerry Shea, Stephen T. Durant, Louise A. Griffiths, Emma E. Fairweather, Kay Eckersley, Dominic I. James, Ian D. Waddell, Donald J. Ogilvie, Paul P. Kelly, Mark J. O'Connor, and Nicola Hamilton
- Subjects
0301 basic medicine ,DNA damage ,DNA repair ,Poly ADP ribose polymerase ,Biology ,DNA damage response ,olaparib ,General Biochemistry, Genetics and Molecular Biology ,PARP ,Olaparib ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Signaling ,Ribose ,PARG ,General Pharmacology, Toxicology and Pharmaceutics ,Poly(ADP-ribose) glycohydrolase ,Base excision repair ,Genetics ,Manchester Cancer Research Centre ,General Immunology and Microbiology ,ResearchInstitutes_Networks_Beacons/mcrc ,Structure: Replication & Repair ,Articles ,General Medicine ,Method Article ,MMS ,030104 developmental biology ,chemistry ,Biochemistry ,ADP ribosylation ,030220 oncology & carcinogenesis ,Medical Genetics - Abstract
After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.
- Published
- 2016
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