Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Rad9/Rad1/Hus1 complex functions to facilitate the ATR-mediated phosphorylation of several substrates that control the checkpoint arrest induced by DNA damage. Here we show that in response to genotoxic stress induced by different types of damaging agents, Rad9 rapidly relocalized to sites of single stranded DNA, as visualized by discrete nuclear foci that co-localize with RPA. UV light-induced Rad9 foci also colocalized with TopBP1 and gamma-H2AX. Interestingly, Rad9 foci were predominately formed in G(1) and S phase after UV light, while treatment of cells with ionizing radiation (IR) resulted in accumulation of Rad9 into foci in S and G(2). Photobleaching experiments in living cells revealed that the Rad9 protein is highly mobile in undamaged cells. However, genotoxic stress induced the immobilization of a large proportion of the protein. The proportion of Rad9 immobilization was larger in S phase and the accumulation to sites of locally damaged areas induced by UV-laser irradiation was faster during DNA replication. Inactivation of nucleotide excision repair by knock down of XPA and XPC resulted in a decrease of G(1) phase cells that displayed Rad9 foci in response to UV light, whereas IR-induced Rad9 foci were not affected. In contrast, downregulation of CtIP, which promotes DSB resection, abrogated the IR-induced Rad9 foci. These findings show that due to processing of DNA lesions into a common intermediate, which occurs in a cell cycle-dependent manner, Rad9 is able to respond to different types of genotoxic stress.
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PMID:Cell cycle-dependent processing of DNA lesions controls localization of Rad9 to sites of genotoxic stress. 1941 45

Maintenance of genome integrity is essential for homeostasis and survival as impaired DNA damage response (DDR) may predispose to grave pathologies such as neurodegenerative and immunodeficiency syndromes, cancer and premature aging. Therefore, accurate assessment of DNA damage caused by environmental or metabolic genotoxic insults is critical for contemporary biomedicine. The available physical, flow cytometry and sophisticated scanning approaches to DNA damage estimation each have some drawbacks such as insufficient sensitivity, limitation to analysis of cells in suspension, or high costs and demand for trained personnel. Here we present an option how to transform a regular fluorescence microscope and personal computer with common software into a functional alternative to high-throughput screening devices. In two detailed protocols we introduce a new semi-automatic procedure allowing for very sensitive, quantitative, rapid and simple fluorescence image analysis in thousands of adherent cells per day. Sensitive DNA breakage estimation through analysis of phosphorylated histone H2AX (gamma-H2AX), and homologous recombination (HR) assessed by a new RPA/Rad51 dual-marker approach illustrate the advantages and applicability of this technique. Our present data on assessment of low radiation doses, repair kinetics, spontaneous DNA damage in cancer cells, as well as constitutive and replication stress-induced HR events and their dependence on upstream factors within the DDR machinery document the versatility of the method. We believe this affordable approach may facilitate mechanistic insights into the role of low-dose DNA damage in human diseases, and generally promote both basic and translational research in many areas of biomedicine where suitable fluorescence markers are available.
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PMID:Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis. 1962 77

The synthetic atypical retinoids containing an adamantyl group exhibit antiproliferative or proapoptotic activities. Apoptosis induction is a dose-dependent effect independent of retinoid receptors. We have reported that induction of apoptosis by the atypical retinoid, ST1926, is associated with early manifestations of genotoxic stress. Indeed, in this study performed in ovarian carcinoma cells, we show that exposure to ST1926 resulted in an increase of early markers of DNA damage, including ATM and H2AX phosphorylation. In addition, we found that a novel histone deacetylase (HDAC) inhibitor (RC307) was able to enhance sensitivity of ovarian carcinoma cells to ST1926. Under conditions where single-agent treatment caused only antiproliferative effects, the combination of the atypical retinoid and HDAC inhibitor resulted in marked apoptotic cell death with a more rapid onset in wild-type p53 ovarian carcinoma cells. The sensitization to ST1926-induced apoptosis was associated with an enhanced DNA damage response, because a prolonged expression of DNA damage markers (e.g., H2AX, p53 and RPA-2 phosphorylation) and a marked activation of DNA damage checkpoint kinases (in particular, phosphorylation of Chk1) were observed indicating an accumulation of DNA damage by the ST1926/HDAC inhibitor combination. The study provides additional support to the role of DNA damage as a primary event leading to the activation of apoptosis in ovarian carcinoma cells by adamantyl retinoids and documents the potential therapeutic efficacy of the combination of ST1926 and HDAC inhibitors of the novel series.
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PMID:Sensitization of ovarian carcinoma cells to the atypical retinoid ST1926 by the histone deacetylase inhibitor, RC307: enhanced DNA damage response. 1967 51

H2AX phosphorylation at serine 139 (gammaH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that gammaH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing gammaH2AX are not committed to death. gammaH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain gammaH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing gammaH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of gammaH2AX foci. Furthermore activated ATM and Chk2 persist in these cells. We propose that the gammaH2AX foci in Chk1-depleted cells may represent sites of persistent replication fork damage or abandonment that are unable to resume DNA synthesis but do not play a direct role in the Chk1 suppressed death pathway.
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PMID:Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1. 2005 81

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55-S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases.
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PMID:A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae. 2006 70

Combined deficiencies of poly(ADP)ribosyl polymerase 1 (PARP1) and ataxia telangiectasia mutated (ATM) result in synthetic lethality and, in the mouse, early embryonic death. Here, we investigated the genetic requirements for this lethality via analysis of mice deficient for PARP1 and either of two ATM-regulated DNA damage response (DDR) factors: histone H2AX and 53BP1. We found that, like ATM, H2AX is essential for viability in a PARP1-deficient background. In contrast, deficiency for 53BP1 modestly exacerbates phenotypes of growth retardation, genomic instability, and organismal radiosensitivity observed in PARP1-deficient mice. To gain mechanistic insights into these different phenotypes, we examined roles for 53BP1 in the repair of replication-associated double-strand breaks (DSBs) in several cellular contexts. We show that 53BP1 is required for DNA-PKcs-dependent repair of hydroxyurea (HU)-induced DSBs but dispensable for RPA/RAD51-dependent DSB repair in the same setting. Moreover, repair of mitomycin C (MMC)-induced DSBs and sister chromatid exchanges (SCEs), two RAD51-dependent processes, are 53BP1 independent. Overall, our findings define 53BP1 as a main facilitator of nonhomologous end joining (NHEJ) during the S phase of the cell cycle, beyond highly specialized lymphocyte rearrangements. These findings have important implications for our understanding of the mechanisms whereby ATM-regulated DDR prevents human aging and cancer.
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PMID:Differential requirement for H2AX and 53BP1 in organismal development and genome maintenance in the absence of poly(ADP)ribosyl polymerase 1. 2023 60

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form gamma-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers.
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PMID:Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2. 2067 34

DNA damage can induce a tumor suppressive response termed cellular senescence. Damaged senescent cells permanently arrest growth, secrete inflammatory cytokines and other proteins and harbor persistent nuclear foci that contain DNA damage response (DDR) proteins. To understand how persistent damage foci differ from transient foci that mark repairable DNA lesions, we identify sequential events that differentiate transient foci from persistent foci, which we term 'DNA segments with chromatin alterations reinforcing senescence' (DNA-SCARS). Unlike transient foci, DNA-SCARS associate with PML nuclear bodies, lack the DNA repair proteins RPA and RAD51, lack single-stranded DNA and DNA synthesis and accumulate activated forms of the DDR mediators CHK2 and p53. DNA-SCARS form independently of p53, pRB and several other checkpoint and repair proteins but require p53 and pRb to trigger the senescence growth arrest. Importantly, depletion of the DNA-SCARS-stabilizing component histone H2AX did not deplete 53BP1 from DNA-SCARS but diminished the presence of MDC1 and activated CHK2. Furthermore, depletion of H2AX reduced both the p53-dependent senescence growth arrest and p53-independent cytokine secretion. DNA-SCARS were also observed following severe damage to multiple human cell types and mouse tissues, suggesting that they can be used in combination with other markers to identify senescent cells. Thus, DNA-SCARS are dynamically formed distinct structures that functionally regulate multiple aspects of the senescent phenotype.
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PMID:DNA-SCARS: distinct nuclear structures that sustain damage-induced senescence growth arrest and inflammatory cytokine secretion. 2111 58

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins.
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PMID:Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM). 2119 12

Numerous anticancer agents and environmental mutagens target DNA. Although all such compounds interfere with the progression of the replication fork and inhibit DNA synthesis, there are marked differences in the DNA-damage response pathways they trigger, and the relative impact of the proximal or the distal signal transducers on cell survival is mainly lesion-specific. Accordingly, checkpoint kinase inhibitors in current clinical development show synergistic activity with some DNA-targeting agents, but not with others. In the present study, we characterize the DNA-damage response to the antitumour acronycine derivative S23906, which forms monofunctional adducts with guanine residues in the minor groove of DNA. S23906 exposure is accompanied by specific recruitment of RPA (replication protein A) at replication sites and rapid Chk1 activation. In contrast, neither MRN (Mre11-Rad50-Nbs1) nor ATM (ataxia-telangiectasia mutated), contributes to the initial response to S23906. Interestingly, genetic attenuation of ATR (ATM- and Ras3-related) activity inhibits not only the early phosphorylation of histone H2AX and Chk1, but also interferes with the late phosphorylation of Chk2. Moreover, loss of ATR function or pharmacological inhibition of the checkpoint kinases by AZD7762 is accompanied by abrogation of the S-phase arrest and increased sensitivity towards S23906. These findings identify ATR as a central co-ordinator of the DNA-damage response to S23906, and provide a mechanistic rationale for combinations of S23906 and similar agents with checkpoint abrogators.
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PMID:Ataxia telangiectasia mutated- and Rad3-related kinase drives both the early and the late DNA-damage response to the monofunctional antitumour alkylator S23906. 2147 Jan 88


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