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An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

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 ye...

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Autores principales: James, Dominic I., Durant, Stephen, Eckersley, Kay, Fairweather, Emma, Griffiths, Louise A., Hamilton, Nicola, Kelly, Paul, O'Connor, Mark, Shea, Kerry, Waddell, Ian D., Ogilvie, Donald J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: F1000Research 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995692/
https://www.ncbi.nlm.nih.gov/pubmed/27610220
http://dx.doi.org/10.12688/f1000research.8463.2
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author James, Dominic I.
Durant, Stephen
Eckersley, Kay
Fairweather, Emma
Griffiths, Louise A.
Hamilton, Nicola
Kelly, Paul
O'Connor, Mark
Shea, Kerry
Waddell, Ian D.
Ogilvie, Donald J.
author_facet James, Dominic I.
Durant, Stephen
Eckersley, Kay
Fairweather, Emma
Griffiths, Louise A.
Hamilton, Nicola
Kelly, Paul
O'Connor, Mark
Shea, Kerry
Waddell, Ian D.
Ogilvie, Donald J.
author_sort James, Dominic I.
collection PubMed
description 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.
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spelling pubmed-49956922016-09-07 An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells James, Dominic I. Durant, Stephen Eckersley, Kay Fairweather, Emma Griffiths, Louise A. Hamilton, Nicola Kelly, Paul O'Connor, Mark Shea, Kerry Waddell, Ian D. Ogilvie, Donald J. F1000Res Method Article 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. F1000Research 2016-09-15 /pmc/articles/PMC4995692/ /pubmed/27610220 http://dx.doi.org/10.12688/f1000research.8463.2 Text en Copyright: © 2016 James DI et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Method Article
James, Dominic I.
Durant, Stephen
Eckersley, Kay
Fairweather, Emma
Griffiths, Louise A.
Hamilton, Nicola
Kelly, Paul
O'Connor, Mark
Shea, Kerry
Waddell, Ian D.
Ogilvie, Donald J.
An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells
title An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells
title_full An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells
title_fullStr An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells
title_full_unstemmed An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells
title_short An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells
title_sort assay to measure poly(adp ribose) glycohydrolase (parg) activity in cells
topic Method Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995692/
https://www.ncbi.nlm.nih.gov/pubmed/27610220
http://dx.doi.org/10.12688/f1000research.8463.2
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