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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX-/- mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.
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PMID:Genomic instability in mice lacking histone H2AX. 1193 88

p53 mutant tumour cells respond to genotoxic insults by bypassing G1 arrest and halting in G2. Following release from G2 arrest they undergo mitotic catastrophe, whereby mitotic cycling is suppressed, delayed apoptosis begins and endopolyploid cells are produced. The ability of these endopolyploid cells to participate in the restitution process is controversial. To facilitate recovery, these endopolyploid cells must repair the extensive DNA damage induced. DNA damage and its resolution were studied by observing the kinetics of gamma-H2AX foci formation and by comet assay analysis. Subsequently, the kinetics and distribution of Rad51 foci were studied as a measure of homologous recombination. Here we present evidence of the resolution of DNA damage in endopolyploid cells through a decrease of tail moment by comet assay and in the number of cells expressing gamma-H2AX foci. Rad51 foci expression reached a maximum in endopolyploid cells on days 5-6 after irradiation, when delayed apoptosis was maximal, indicating that cells were being selected for survival at this time. Furthermore, the proportion of Annexin-V-positive polyploid cells decreased as they continued ongoing rounds of DNA replication, suggesting endoreduplication is involved in selecting cells resistant to apoptosis. Our findings suggest that after severe genotoxic insult endopolyploid cells have a transient survival advantage that may contribute to radioresistance of tumours that undergo mitotic catastrophe.
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PMID:Endopolyploid cells produced after severe genotoxic damage have the potential to repair DNA double strand breaks. 1295 71

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms
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PMID:Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells. 1296 Apr 8

The temporal and functional relationships between DNA events of meiotic recombination and synaptonemal complex formation are a matter of discussion within the meiotic field. To analyse this subject in grasshoppers, organisms that have been considered as models for meiotic studies for many years, we have studied the localization of phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks (DSBs), in combination with localization of cohesin SMC3 and recombinase Rad51. We show that the loss of gamma-H2AX staining is spatially and temporally linked to synapsis, and that in grasshoppers the initiation of recombination, produced as a consequence of DSB formation, precedes synapsis. This result supports the idea that grasshoppers display a pairing pathway that is not present in other insects such as Drosophila melanogaster, but is similar to those reported in yeast, mouse and Arabidopsis. In addition, we have observed the presence of gamma-H2AX in the X chromosome from zygotene to late pachytene, indicating that the function of H2AX phosphorylation during grasshopper spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA DSBs.
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PMID:DNA double-strand breaks, recombination and synapsis: the timing of meiosis differs in grasshoppers and flies. 1510 29

We have analysed the chromosome organisation and the location and temporal appearance of different proteins in X and B chromosomes in the grasshopper Eyprepocnemis plorans throughout the first meiotic prophase. We have used adult males that carry a B chromosome collected in natural Spanish populations. The scaffold organisation has been analysed by means of silver stained chromatid cores. In addition, we have detected by immunolabelling the presence of phosphoepitopes, the ensemble of cohesin axes, the location of histone gamma-H2AX, and recombinase Rad51. Our observations demonstrate that X and B chromosomes share similarities in chromatin organisation and in the expression of the tested proteins, which strongly differ from those of the autosomes. These results could be interpreted either as a support to the hypothesis that the Bs analysed here originated from the X chromosome, and/or that their chromatin composition and precocious condensation could determine their meiotic behaviour.
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PMID:X and B chromosomes display similar meiotic characteristics in male grasshoppers. 1529 7

After induction of DNA double-strand breaks (DSB) two repair systems, the error-prone 'nonhomologous end joining' (NHEJ) and the more accurate 'homologous recombination repair' (HRR) can compete for the same individual DSB site. In the human keratinocyte cell line, HaCaT, we have tested the spatial co-localisation and the temporal sequence of events. We used UV-A (365 nm) as a damaging agent, which can be applied in clearly defined doses and can lead to rare DSBs via propagation of clustered single-strand breaks (SSBs). DNA fragmentation and repair was measured by the Comet assay and persisting DSBs were quantified by the micronucleus assay. Direct DSB detection was performed by immunohistochemical labelling of gamma-H2AX, a phosphorylated histone that is assumed to form one foci per DSB. Intra- and inter-pathway interactions were quantified by co-localisation, FRET imaging and by co-immunoprecipitation (Co-IP) of XRCC4, DNA-PK and Ku70 as representatives of NHEJ, Rad51 and Rad52 for HRR and gamma-H2AX, Mre11 and Rad50 as representatives of both pathways. In G2 cells, where both systems are available, the temporal sequence after irradiation is: (1) gamma-H2AX (2) Mre11 (3) DNA-PK Rad51 (4) XRCC4. That is, the first two proteins involved in both pathways 'label' the damaged site and initiate repair, followed by the NHEJ, which is temporally overlapping with HRR activity. Taking all these observations together we suggest that a cell tries to repair DSBs with a combination of both HRR and NHEJ, if available.
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PMID:After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available. 1536 81

Histone H2AX has a role in suppressing genomic instability and cancer. However, the mechanisms by which it performs these functions are poorly understood. After DNA breakage, H2AX is phosphorylated on serine 139 in chromatin near the break. We show here that H2AX serine 139 enforces efficient homologous recombinational repair of a chromosomal double-strand break (DSB) by using the sister chromatid as a template. BRCA1, Rad51, and CHK2 contribute to recombinational repair, in part independently of H2AX. H2AX(-/-) cells show increased use of single-strand annealing, an error-prone deletional mechanism of DSB repair. Therefore, the chromatin response around a chromosomal DSB, in which H2AX serine 139 phosphorylation plays a central role, "shapes" the repair process in favor of potentially error-free interchromatid homologous recombination at the expense of error-prone repair. H2AX phosphorylation may help set up a favorable disposition between sister chromatids.
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PMID:Control of sister chromatid recombination by histone H2AX. 1561 Jul 43

DNA polymerase (Pol) beta null mouse embryonic fibroblasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are characterized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol beta, SSB accumulate in G1 phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol beta null cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are substrates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity.
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PMID:The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells. 1564 10

The aim of the present work was to study the role of Rad51-dependent homologous recombination in the radiation response of non-small-cell lung cancer (NSCLC) cell lines. A dose- and time-dependent increase in the formation of Rad51 and gamma-H2AX foci with a maximum at about 4 and 1 h after irradiation, followed by a decrease, has been found. The relative fraction of cells with persisting Rad51 foci was 20-30% in radioresistant and 60-80% in radiosensitive cell lines. In comparison, a higher fraction of residual Dsb was evident in cell lines with nonfunctional p53. Transfection with As-Rad51 significantly downregulates radiation-induced formation of Rad51 foci and increases apoptosis, but did not influence the rejoining of DNA double-strand breaks. Interestingly, wortmannin, a well-known inhibitor of nonhomologous end-joining, also inhibits Rad51 foci formation. In general, there was no correlation between the clonogenic survival at 2 Gy and the percentage of initial Rad51 or gamma-H2AX foci after ionising radiation (IR). The most reliable predictive factor for radiosensitivity of NSCLC cell lines was the relative fraction of Rad51 foci remaining at 24 h after IR. Although most of the Rad51 foci are co-localised with gamma-H2AX foci, no correlation of the relative fraction of persisting gamma-H2AX foci and SF2 is evident.
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PMID:Targeting of Rad51-dependent homologous recombination: implications for the radiation sensitivity of human lung cancer cell lines. 1578 36

Induction of apoptosis is pivotal for eliminating cells with damaged DNA or deregulated proliferation. We show that tumor suppressor ARF and ATM/ATR kinase pathways cooperate in the induction of apoptosis in response to elevated expression of c-myc, beta-catenin or human papilloma virus E7 oncogenes. Overexpression of oncogenes leads to the formation of phosphorylated H2AX foci, induction of Rad51 protein levels and ATM/ATR-dependent phosphorylation of p53. Inhibition of ATM/ATR kinases abolishes both induction of Rad51 and phosphorylation of p53, and remarkably reduces the level of apoptosis induced by co-expression of oncogenes and ARF. However, the induction of apoptosis is downregulated in p53-/- cells and does not depend on activities of ATM/ATR kinases, indicating that efficient induction of apoptosis by oncogene activation depends on coordinated action of ARF and ATM/ATR pathways in the regulation of p53.
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PMID:ARF and ATM/ATR cooperate in p53-mediated apoptosis upon oncogenic stress. 1600 68


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