Analysis of DNA damage in individual cells

Olive, Peggy L.; Durand, Ralph E.; Banáth, Judit P.; Johnston, Peter J.

doi:10.1016/s0091-679x(01)64016-0

Cited by 67 publications

(34 citation statements)

References 42 publications

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“…To confirm that Iduna facilitates DNA repair, the alkaline comet assay was performed. The comet assay detects DNA fragmentation by monitoring DNA integrity by SYBR green staining during electrophoresis of cells (20). Cells with intact DNA have compact circular staining, whereas cells with DNA damage have bright tails that resemble comets.…”

Section: Resultsmentioning

confidence: 99%

Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

Kang

Lee

Shin

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

229

221

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Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair.

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Section: Resultsmentioning

confidence: 99%

Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

Kang

Lee

Shin

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

229

221

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“…While approaches to measure the presence of oxidants within the cell have been developed, 10,11 the possibilities for detecting DSBs generated by endogenous oxidants are limited. DNA damage assessment by the comet methodology 12 is rather cumbersome, lacks the desired sensitivity and cannot provide information on the relationship between DNA damage and the cell cycle phase in which the damage occurred. The detection of DSBs by the TUNEL assay, while highly useful in identifying DNA fragmentation that occurs during apoptosis, 13 also is not sensitive enough to detect DNA damage induced by endogenous oxidants.…”

mentioning

confidence: 99%

Constitutive Histone H2AX Phosphorylation and ATM Activation, the Reporters of DNA Damage by Endogenous Oxidants

Tanaka

Halicka²,

Huang³

et al. 2006

Cell Cycle

190

197

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DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of histone H2AX on Ser 139; the phosphorylated form of H2AX has been named γH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS. Keywordsaging; senescence; DNA replication; DNA double-strand breaks; cell cycle; free radicals; reactive oxygen species (ROS); reactive oxygen intermediates (ROIs); anti-oxidants; oxidative stress DNA DAMAGE BY ENDOGENOUS OXIDANTSBeing continuously exposed to oxidants produced during metabolic activity and to external oxidants or oxidant-inducers, DNA within cells undergoes oxidative damage. Estimates of the extent of endogenous DNA damage vary widely. [1][2][3][4][5] 5 Recombinatorial repair (also known as template-assisted repair or homologous recombination repair) and nonhomologous DNA-end joining (NHEJ) are two major pathways for repair of DSBs. The NHEJ pathway is error-prone, often resulting in deletion of a few base pairs. 6,7 This leads to accumulation of DNA damage with each sequential cell cycle, which is considered to be the primary cause of cell aging and senescence. 4,8,9 The cumulative DNA damage also promotes development of preneoplastic changes. Many strategies aimed at slowing down the aging process or preventing cancer are based on protection of DNA from oxidative damage, primarily by scavenging the endogenous oxidants. While appr...

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“…Scored parameters included tail length, head DNA % and Olive tail moment. The Olive tail moment is a global comet parameter expressed as [(tail mean -head mean) × (% tail DNA/100)] and used to quantify DNA damage [Olive et al, 2001].…”

Section: Comet Assaymentioning

confidence: 99%

Evaluation of Mango Byproduct Extracts as Antioxidant against Pb-acetate induced Oxidative Stress and Genotoxicity in Mice

Makawy¹,

Ashoush²,

Abd-El-Moneim³

2015

Pol. J. Food Nutr. Sci.

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The antioxidant and antiproliferative properties of mango by-products were investigated. This study was carried out to evaluate the protective role of mango peel or kernel defatted extracts against Pb-acetate adverse effects on oxidant/antioxidant status, liver dysfunction biomarkers, histopathological changes and genotoxicity in male mice. Total phenolic content and antioxidant activity of both extracts were evaluated. Two doses of both extracts (50 and 100 mg/kg) were used to evaluate their role against the toxicity of Pb-acetate (500 ppm). Mice given mango extracts with Pb-acetate had signifi cantly lower plasma MDA, AST and ALT and higher glutathione than mice given Pb-acetate alone. Mango extracts prevented the histopathological changes in liver induced by Pb-acetate and decreased the cytotoxicity of lead by increasing the ratio of PCE/NCE. Mango extract treatment reduced the DNA damage induced by Pb-acetate in liver as demonstrated by a reduction in micronuclei and decrease in tail length, tail DNA% and Olive tail moment. It can be concluded that mango by-product extracts have potential to protect from oxidative stress and genotoxicity of lead.Unauthenticated Download Date | 5/8/18 7:04 AM

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Analysis of DNA damage in individual cells

Cited by 67 publications

References 42 publications

Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

Constitutive Histone H2AX Phosphorylation and ATM Activation, the Reporters of DNA Damage by Endogenous Oxidants

Evaluation of Mango Byproduct Extracts as Antioxidant against Pb-acetate induced Oxidative Stress and Genotoxicity in Mice

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