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)

H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.
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PMID:ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. 1505 90

To investigate the repair of clustered lesions within the DNA/chromatin, the focus formation and persistence of foci of the phosphorylated histone protein H2AX and the repair protein MRE11 were studied in normal cells and in cells lacking DNA-PKcs (M059J) or ATM (GM2052D) after irradiation with high-LET nitrogen ions or low-LET photons. There was a rapid formation of MRE11 and gamma-H2AX foci, and 0.5 h after high-LET irradiation, the number of foci in normal cells correlated well with the number of particle hits per cell nucleus. After 8 h of repair, there were significantly more gamma-H2AX foci than MRE11 foci remaining in the normal cells, independent of radiation quality. The difficulty in repairing clustered breaks was detected as slower rejoining of DSBs (measured by DNA fragmentation analysis), as quantification of the amount of gamma-H2AX over time, and as a larger fraction of repair foci remaining after 24 h in cells irradiated with high- LET ions. These data indicate that clustered lesions are repaired by a pathway involving the same proteins that repair sparsely distributed breaks. Further, for both low- and high- LET radiation, no reduction of the initial number of gamma-H2AX and MRE11 foci was detected in M059J cells up to 21 h after irradiation, which was in accordance with a complete absence of DSB rejoining in these cells. In the GM2052D cells there was also a higher level of foci remaining after 21 h; however, this was not accompanied by unrejoined DSBs, indicating that these foci not only represent DSBs but also may be a sign of persistent problems even when breaks are rejoined.
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PMID:Focus formation of DNA repair proteins in normal and repair-deficient cells irradiated with high-LET ions. 1516 72

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

In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.
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PMID:Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors. 1538 85

The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break repair (DSBR) proteins. Most of the known activities of E4orf6 during adenoviral infection require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6, stably expressed in RKO human colorectal carcinoma cells or transiently expressed by adenoviral vector in U251 human glioblastoma cells, inhibits DSBR and induces significant radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P) failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (gamma-H2AX) levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid (pSP189) in 293 cells but did not increase the mutation frequency measured in the reporter plasmid. The kinase activity of DNA-PKcs (the DNA-dependent protein kinase catalytic subunit) toward heterologous substrates was not affected by expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr-2609 following ionizing radiation was prolonged in the presence of E4orf6 when compared with control-infected cells. Our results demonstrate for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian cells in the absence of E1B-55K or other adenoviral genes and suggest that viral-mediated delivery of E4orf6, combined with localized external beam radiation, could be a useful approach for the treatment of radioresistant solid tumors such as glioblastomas.
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PMID:The adenovirus E4orf6 protein inhibits DNA double strand break repair and radiosensitizes human tumor cells in an E1B-55K-independent manner. 1550 30

Repair of DNA double strand breaks (DSBs) by the non-homologous end joining (NHEJ) pathway in mammals requires at least the DNA-dependent protein kinase (DNA-PK) and the DNA ligase IV-XRCC4 protein complexes. DNA-PK comprises the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs. Here we report the first description of the nuclear mobilization of endogenous NHEJ proteins after exposure of human cells to double strand-breaking agents. DSB infliction specifically induced a dose- and time-dependent mobilization of Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV proteins from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. XRCC4 recruitment was accompanied by its DNA-PK-dependent phosphorylation. The recruited proteins co-immunoprecipitated, indicating that they had assembled into complexes. However, DNA-PK was attached to chromatin, whereas XRCC4-ligase IV resisted solubilization by DNase I. The rates of appearance and dissolution of NHEJ proteins paralleled that of histone variant H2AX phosphorylation and dephosphorylation. We established that under conditions of genomic DSB infliction 1) Ku recruitment was not dependent on the co-recruitment of the other NHEJ proteins, 2) DNA-PKcs was physically required for the mobilization of the XRCC4-ligase IV complex, 3) DNA ligase IV was physically necessary for stable recruitment of XRCC4, and 4) phosphorylation of either H2AX or XRCC4 was unnecessary for DNA-PK or XRCC4-ligase IV recruitment. Altogether these results offer insights into the interplay between key NHEJ proteins during this repair process in the cell.
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PMID:DNA-dependent protein kinase and XRCC4-DNA ligase IV mobilization in the cell in response to DNA double strand breaks. 1552 13

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.
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PMID:A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci. 1557 27

It has been established that telomere-dependent replicative senescence of human fibroblasts is stress-dependent. First, it was shown that telomere shortening, which is a major contributor to telomere uncapping, is stress-dependent to a significant degree. Second, the signalling pathway connecting telomere uncapping and replicative senescence appears to be the same as the one that is activated by DNA damage: uncapped telomeres activate signalling cascades involving the protein kinases ATM, ATR and, possibly, DNA-PK. Furthermore, phosphorylation of histone H2A.X facilitates the formation of DNA damage foci around uncapped telomeres, and this in turn activates downstream kinases Chk1 and Chk2 and, eventually, p53. It appears that this signalling pathway has to be maintained in order to keep cells in a senescent state. Thus, cellular senescence can be regarded as a permanently maintained DNA damage response state. This suggests that antibodies against DNA damage foci components might be useful markers for senescent cells in vivo.
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PMID:Human cell senescence as a DNA damage response. 1561 Jul 69

Formation of gamma-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that gamma-H2AX foci were also formed when cells were incubated with 0.5 microg/ml DNA intercalating agent actinomycin D. In untreated cells, gamma-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with gamma-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and gamma-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to gamma-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with gamma-H2AX foci. Taken together, these results suggest that histone gamma-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA.
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PMID:Actinomycin D induces histone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70 and nuclear DNA helicase II. 1561 78

Retroviral DNA integration creates a discontinuity in the host cell chromatin and repair of this damage is required to complete the integration process. As integration and repair are essential for both viral replication and cell survival, it is possible that specific interactions with the host DNA repair systems might provide new cellular targets for human immunodeficiency virus therapy. Various genetic, pharmacological, and biochemical studies have provided strong evidence that postintegration DNA repair depends on components of the nonhomologous end-joining (NHEJ) pathway (DNA-PK (DNA-dependent protein kinase), Ku, Xrcc4, DNA ligase IV) and DNA damage-sensing pathways (Atr (Atm and Rad related), gamma-H2AX). Furthermore, deficiencies in NHEJ components result in susceptibility to apoptotic cell death following retroviral infection. Here, we review these findings and discuss other ways that retroviral DNA intermediates may interact with the host DNA damage signaling and repair pathways.
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PMID:Retroviral DNA integration and the DNA damage response. 1576 74


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