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

Dominic I James; Stephen Durant; Kay Eckersley; Emma Fairweather; Louise A Griffiths; Nicola Hamilton; Paul Kelly; Mark O'Connor; Kerry Shea; Ian D Waddell; Donald J Ogilvie

doi:10.12688/f1000research.8463.2

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

Dominic I James, Stephen Durant, Kay Eckersley, Emma Fairweather, Louise A Griffiths, Nicola Hamilton, Paul Kelly, Mark O'Connor, Kerry Shea, Ian D Waddell, Donald J Ogilvie

Cancer Research UK Manchester Institute

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Pages (from-to)	736
Journal	F1000Research
Volume	5
Issue number	736
DOIs	https://doi.org/10.12688/f1000research.8463.2
Publication status	Published - 25 Apr 2016

Keywords

PARG,

Research Beacons, Institutes and Platforms

Manchester Cancer Research Centre

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.12688/f1000research.8463.2Licence: CC BY

Cite this

@article{528371f1783a45c2b2891410150ba408,

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.",

keywords = "PARG, ",

author = "James, {Dominic I} and Stephen Durant and Kay Eckersley and Emma Fairweather and Griffiths, {Louise A} and Nicola Hamilton and Paul Kelly and Mark O'Connor and Kerry Shea and Waddell, {Ian D} and Ogilvie, {Donald J}",

year = "2016",

month = apr,

day = "25",

doi = "10.12688/f1000research.8463.2",

language = "English",

volume = "5",

pages = "736",

journal = "F1000Research",

issn = "2046-1402",

publisher = "Faculty of 1000 Ltd",

number = "736",

}

TY - JOUR

T1 - An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

AU - James, Dominic I

AU - Durant, Stephen

AU - Eckersley, Kay

AU - Fairweather, Emma

AU - Griffiths, Louise A

AU - Hamilton, Nicola

AU - Kelly, Paul

AU - O'Connor, Mark

AU - Shea, Kerry

AU - Waddell, Ian D

AU - Ogilvie, Donald J

PY - 2016/4/25

Y1 - 2016/4/25

N2 - 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.

AB - 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.

KW - PARG,

U2 - 10.12688/f1000research.8463.2

DO - 10.12688/f1000research.8463.2

M3 - Article

C2 - 27610220

SN - 2046-1402

VL - 5

SP - 736

JO - F1000Research

JF - F1000Research

IS - 736

ER -

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

Abstract

Keywords

Research Beacons, Institutes and Platforms

UN SDGs

Access to Document

Fingerprint

Cite this