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)

MRE11, RAD50 and NBS1 form a highly conserved protein complex (the MRE11 complex) that is involved in the detection, signalling and repair of DNA damage. We identify MDC1 (KIAA0170/NFBD1), a protein that contains a forkhead-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains, as a binding partner for the MRE11 complex. We show that, in response to ionizing radiation, MDC1 is hyperphosphorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear foci that also contain the MRE11 complex, phosphorylated histone H2AX and 53BP1. Downregulation of MDC1 expression by small interfering RNA yields a radio-resistant DNA synthesis (RDS) phenotype and prevents ionizing radiation-induced focus formation by the MRE11 complex. However, downregulation of MDC1 does not abolish the ionizing radiation-induced phosphorylation of NBS1, CHK2 and SMC1, or the degradation of CDC25A. Furthermore, we show that overexpression of the MDC1 FHA domain interferes with focus formation by MDC1 itself and by the MRE11 complex, and induces an RDS phenotype. These findings reveal that MDC1-mediated focus formation by the MRE11 complex at sites of DNA damage is crucial for the efficient activation of the intra-S-phase checkpoint.
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PMID:MDC1 is required for the intra-S-phase DNA damage checkpoint. 1260 3

To counteract the continuous exposure of cells to agents that damage DNA, cells have evolved complex regulatory networks called checkpoints to sense DNA damage and coordinate DNA replication, cell-cycle arrest and DNA repair. It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage. Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints. MDC1 forms foci that co-localize extensively with gamma-H2AX foci within minutes after exposure to ionizing radiation. H2AX is required for MDC1 foci formation, and MDC1 forms complexes with phosphorylated H2AX. Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner. We have shown by using small interfering RNA (siRNA) that cells lacking MDC1 are sensitive to ionizing radiation, and that MDC1 controls the formation of damage-induced 53BP1, BRCA1 and MRN foci, in part by promoting efficient H2AX phosphorylation. In addition, cells lacking MDC1 also fail to activate the intra-S phase and G2/M phase cell-cycle checkpoints properly after exposure to ionizing radiation, which was associated with an inability to regulate Chk1 properly. These results highlight a crucial role for MDC1 in mediating transduction of the DNA damage signal.
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PMID:MDC1 is a mediator of the mammalian DNA damage checkpoint. 1260 5

Ionizing radiation induces genomic instability, which is transmitted through many generations after irradiation in the progeny of surviving cells. To detect delayed activation of p53, we constructed a reporter plasmid containing the p53-responsible promoter and the bacterial beta-galactosidase (beta-gal) gene and introduced it into human fibrosarcoma (HT1080) cells, which retain wild-type p53 function. The resultant clones induce beta-gal protein after X-irradiation, and the induction kinetics were similar to those of p21(WAF1/CIP1) protein. More than 90% of the cells were stained blue when the cells were incubated with X-gal 4 h after 6 Gy of X-rays, whereas very few control cells were beta-gal positive. The primary colonies formed after 6 Gy of X-rays were collected, and they were subjected to secondary colony formation. We observed that a significant number of surviving colonies contained beta-gal-positive cells, suggesting that delayed activation of p53 occurred in the progeny of irradiated cells. We also found higher frequency of phosphorylation of p53, NBS1, and CHK2/Cds1 in the progeny of surviving cells. Furthermore, foci formation of phosphorylated histone H2AX was detected in the progeny of surviving cells. These findings provide the possibility that the observed instability results from these DNA breaks, i.e., the breaks lead to delayed chromosome rearrangements, delayed cell death, and so forth, many generations after irradiation and that activation of p53 function may eliminate cells that have potentially accumulated genomic alterations.
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PMID:Delayed reactivation of p53 in the progeny of cells surviving ionizing radiation. 1261 6

It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (gamma-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/ approximately kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.
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PMID:Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes. 1262 Sep 38

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci. Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This gammaH2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form gammaH2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision. gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.
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PMID:Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. 1266 Feb 52

DNA double-strand breaks (DSBs) are generally accepted to be the most biologically significant lesion by which ionizing radiation causes cancer and hereditary disease. However, no information on the induction and processing of DSBs after physiologically relevant radiation doses is available. Many of the methods used to measure DSB repair inadvertently introduce this form of damage as part of the methodology, and hence are limited in their sensitivity. Here we present evidence that foci of gamma-H2AX (a phosphorylated histone), detected by immunofluorescence, are quantitatively the same as DSBs and are capable of quantifying the repair of individual DSBs. This finding allows the investigation of DSB repair after radiation doses as low as 1 mGy, an improvement by several orders of magnitude over current methods. Surprisingly, DSBs induced in cultures of nondividing primary human fibroblasts by very low radiation doses (approximately 1 mGy) remain unrepaired for many days, in strong contrast to efficient DSB repair that is observed at higher doses. However, the level of DSBs in irradiated cultures decreases to that of unirradiated cell cultures if the cells are allowed to proliferate after irradiation, and we present evidence that this effect may be caused by an elimination of the cells carrying unrepaired DSBs. The results presented are in contrast to current models of risk assessment that assume that cellular responses are equally efficient at low and high doses, and provide the opportunity to employ gamma-H2AX foci formation as a direct biomarker for human exposure to low quantities of ionizing radiation.
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PMID:Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. 1270 28

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2 phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.
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PMID:High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair. 1268 26

During meiotic prophase in male mammals, the X and Y chromosomes condense to form a macrochromatin body, termed the sex, or XY, body, within which X- and Y-linked genes are transcriptionally repressed. The molecular basis and biological function of both sex body formation and meiotic sex chromosome inactivation (MSCI) are unknown. A phosphorylated form of H2AX, a histone H2A variant implicated in DNA repair, accumulates in the sex body in a manner independent of meiotic recombination-associated double-strand breaks. Here we show that the X and Y chromosomes of histone H2AX-deficient spermatocytes fail to condense to form a sex body, do not initiate MSCI, and exhibit severe defects in meiotic pairing. Moreover, other sex body proteins, including macroH2A1.2 and XMR, do not preferentially localize with the sex chromosomes in the absence of H2AX. Thus, H2AX is required for the chromatin remodeling and associated silencing in male meiosis.
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PMID:H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis. 1268 89

Acidic pH plays an important role in various pathophysiological states and has been demonstrated to be carcinogenic in animal models. Recent studies have also implicated acidic pH in the development of preneoplastic Barrett's esophagus in human. However, little is known about the molecular mechanism underlying acidic pH-induced carcinogenesis. In the current study, we show that acidic pH, like the topoisomerase II (TOP2) poison VP-16 (demethylepipodophyllotoxin ethylidene-beta-D-glucoside), induces tumors in 9,10-dimethyl-1,2-benzanthracene(DMBA)-initiated mice. The following studies in tissue culture models have suggested that acidic pH acts like a TOP2 poison to induce TOP2-mediated DNA damage: (i) acidic pH induces TOP2-dependent DNA damage signals as evidenced by up-regulation of p53 and Ser-139 phosphorylation of H2AX [a substrate for ataxia telangiectasia mutated (ATM)ATM and Rad3-related (ATR) kinases]; (ii) acidic pH-induced cytotoxicity in tumor cells is reduced in TOP2-deficient cells; (iii) acidic pH increases the mutation frequency of the hypoxanthine phosphoribosyl transferase (HPRT) gene in a TOP2-dependent manner; and (iv) acidic pH induces reversible TOP2-mediated DNA strand breaks in vitro. We discuss the possibility that TOP2-mediated DNA damage may contribute to acidic pH-induced carcinogenesis.
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PMID:Acidic pH induces topoisomerase II-mediated DNA damage. 1269 9

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.
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PMID:Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. 1269 68


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