title: An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.
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.
authors.affiliation: Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.
authors.forename: Dominic I
authors.initials: DI
authors.lastname: James
authors.affiliation: Oncology iMED, AstraZeneca Pharmaceuticals, Macclesfield, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Durant
authors.affiliation: Oncology iMED, AstraZeneca Pharmaceuticals, Macclesfield, UK.
authors.forename: Kay
authors.initials: K
authors.lastname: Eckersley
authors.affiliation: Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Fairweather
authors.affiliation: Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.
authors.forename: Louise A
authors.initials: LA
authors.lastname: Griffiths
authors.affiliation: Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Hamilton
authors.affiliation: Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Kelly
authors.affiliation: Oncology iMED, AstraZeneca Pharmaceuticals, Macclesfield, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: O'Connor
authors.affiliation: Oncology iMED, AstraZeneca Pharmaceuticals, Macclesfield, UK.
authors.forename: Kerry
authors.initials: K
authors.lastname: Shea
authors.affiliation: Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.
authors.forename: Ian D
authors.initials: ID
authors.lastname: Waddell
authors.affiliation: Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Ogilvie
keywords: ADP ribosylation; Base excision repair; DNA damage response; MMS; PARG; PARP; olaparib