γH2Ax: Biomarker of Damage or Functional Participant in DNA Repair “All that Glitters Is not Gold!”

Cleaver, James E.

doi:10.1111/j.1751-1097.2011.00995.x

Cited by 101 publications

(89 citation statements)

References 57 publications

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“…In agreement with earlier observations (31,33) immunofluorescence revealed that the cell distribution of γH2AX at each time point was not homogeneous (Fig S3). In a parallel experiment, flow cytometry demonstrated greater heterogeneity of the fluorescent signal in mutant fibroblasts than in control fibroblasts during the 24-h repair period (Fig.…”

Section: Resultssupporting

confidence: 80%

“…DNA damage by UV also induces γH2AX in both proliferating and quiescent cells (31,32), but its relevance to this connection is unclear. Thus, it was proposed that γH2AX might act as a biomarker of UV damage rather than as a participant in repair (33). During NER, the presence of γH2AX signals a persistence of DNA gaps created by excision of the UV-induced damage, and its disappearance signals filling of the gaps by dNTP polymerization.…”

Section: Resultsmentioning

confidence: 99%

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Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Pontarin

Ferraro

Bee

et al. 2012

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

114

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In postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage.DNA precursors | dNTP de novo synthesis | cell cycle | mitochondrial disease D NA replication and repair require the continued synthesis of the four dNTPs. They are synthesized by evolutionary-related ribonucleotide reductases operating with slightly different mechanisms in aerobic and anaerobic organisms (1). Each ribonucleotide reductase provides the required amounts of all four dNTPs. A similar allosteric mechanism, maintained throughout evolution, regulates both the enzyme's activity and its substrate specificity. Cells contain small dNTP pools of similar sizes, approximately 10-fold larger during DNA replication than during quiescence. Regulation of pool sizes by ribonucleotide reductases is of great importance for correct DNA replication, and changes in the actual sizes or in their balance lead to increased mutation rates (2). For mammalian cells, the induction of mutations by pool imbalances has been described in detail, along with possible mechanisms (3). In yeast, a recent elegant study (4) linked specific amino acid substitutions in the catalytic subunit of ribonucleotide reductase to defined pool imbalances, which result in increased mutation rates.In mammalian cells, the canonical ribonucleotide reductase is a complex between two proteins: the large catalytic protein R1 that contains the allosteric sites and the smaller protein R2 that contributes a stable tyrosyl free radical during the reaction (1). Both proteins are transcriptionally activated during early S-phase (5) a...

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Pontarin

Ferraro

Bee

et al. 2012

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

114

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“…An anti-cH2AX antibody was used to further analyze the DNA damage in infected cells; however, high-background caused by scattered noise and sporadic, undetermined pan-nuclear staining was observed for both infected and control cells, making the foci unable to be quantified in the microscope. This is in agreement with Cleaver (Cleaver, 2011) who showed that cH2AX plays roles in other cellular events and is not a specific marker for DNA damage. However, increased fluorescent signals upon cH2AX staining was clearly visible in infected cells as well as a clear co-localization with the 53BP1-positive foci (Fig.…”

Section: The Expression Of 53bp1 Foci Was Increased During Gonococcalsupporting

confidence: 81%

Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells

Vielfort

Söderholm

Weyler

et al. 2013

Journal of Cell Science

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SummaryThe constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (cH2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

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“…36,37 As expected, transiently transfected PARP1-GFP was concentrated in the nucleoli of WT MEFs under normal conditions and migrated to the nucleoplasm after exposure to H 2 O 2 ( Figures 5a and b) or UV radiation (Figure 5b). Although the DNA damage was comparable in UVirradiated WT and Ulk1-KO MEFs, as suggested by the similar levels of γH2AX (a biomarker of DNA damage) 38 up to 4 h after treatment (Supplementary Figure S4), PARP1-GFP was retained in the nucleoli of Ulk1-KO MEFs exposed to UV radiation or H 2 O 2 (Figures 5a and b). Next, we performed fluorescence recovery after photobleaching experiments to determine whether PARP1 mobility was altered by ULK1.…”

mentioning

confidence: 68%

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

et al. 2015

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Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H 2 O 2 ). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H 2 O 2 -treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions. Cell Death and Differentiation (2016) 23, 216-230; doi:10.1038/cdd.2015; published online 3 July 2015Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H 2 O 2 ), are formed by the incomplete reduction of oxygen during oxidative phosphorylation and other enzymatic processes. ROS are signaling molecules that regulate cell proliferation, differentiation, and survival. 1-3 Accumulation of ROS (i.e., oxidative stress) on exposure to xenobiotic agents or environmental toxins can cause cellular damage and death via apoptotic or nonapoptotic pathways. [4][5][6] Oxidative stress-induced cellular damage and death have been implicated in aging, ischemia-reperfusion injury, inflammation, and the pathogenesis of diseases (e.g., neurodegeneration and cancer). 7 Oxidative stress also contributes to the antitumor effects of many chemotherapeutic drugs, including camptothecin 8,9 and selenite. 10,11 Autophagy, an evolutionarily conserved intracellular catabolic process, involves lysosome-dependent degradation of superfluous and damaged cytosolic organelles and proteins. 12 It is typically upregulated under conditions of perceived stress and in response to cellular damage. The consequence of autophagy activation -whether cytoprotective or cytotoxicappears to depend on the cell type and the nature and extent of stress. Although most studies indicate a cytoprotective role for autophagy, some evidence suggests that it contributes to cell death in response to oxidative stress. [13][14][15][16][17] Studies have also indicated that autophagy may be suppressed in response to oxidative stress, thereby sensitizing certain cells to apoptosis. 18,19 Unc-51-like kinase/autophagy 1 (ULK1/ATG1) is a mammalian serine-threonine kinase that regulates flux through the autophagy pathway by activating the VPS34 PI(3) kinas...

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γH2Ax: Biomarker of Damage or Functional Participant in DNA Repair “All that Glitters Is not Gold!”

Cited by 101 publications

References 57 publications

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

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