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)

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.
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PMID:ATM phosphorylates histone H2AX in response to DNA double-strand breaks. 1157 Dec 74

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.
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PMID:Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis. 1180 72

V(D)J recombination is the specialized DNA rearrangement used by cells of the immune system to assemble immunoglobulin and T-cell receptor genes from the preexisting gene segments. Because there is a large choice of segments to join, this process accounts for much of the diversity of the immune response. Recombination is initiated by the lymphoid-specific RAG1 and RAG2 proteins, which cooperate to make double-strand breaks at specific recognition sequences (recombination signal sequences, RSSs). The neighboring coding DNA is converted to a hairpin during breakage. Broken ends are then processed and joined with the help of several factors also involved in repair of radiation-damaged DNA, including the DNA-dependent protein kinase (DNA-PK) and the Ku, Artemis, DNA ligase IV, and Xrcc4 proteins, and possibly histone H2AX and the Mre11/Rad50/Nbs1 complex. There may be other factors not yet known. V(D)J recombination is strongly regulated by limiting access to RSS sites within chromatin, so that particular sites are available only in certain cell types and developmental stages. The roles of enhancers, histone acetylation, and chromatin remodeling factors in controlling accessibility are discussed. The RAG proteins are also capable of transposing RSS-ended fragments into new DNA sites. This transposition helps to explain the mechanism of RAG action and supports earlier proposals that V(D)J recombination evolved from an ancient mobile DNA element.
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PMID:V(D)J recombination: RAG proteins, repair factors, and regulation. 1204 92

Nonhomologous end-joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PK catalytic subunit (DNA-PKcs), is activated by DNA in vitro and is required for NHEJ. We report that DNA-PKcs is autophosphorylated at Thr2609 in vivo in a Ku-dependent manner in response to ionizing radiation. Phosphorylated DNA-PKcs colocalizes with both gamma-H2AX and 53BP1 after DNA damage. Mutation of Thr2609 to Ala leads to radiation sensitivity and impaired DSB rejoining. These findings establish that Ku-dependent phosphorylation of DNA-PKcs at Thr2609 is required for the repair of DSBs by NHEJ.
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PMID:Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. 1223 22

Ultraviolet (UV) irradiation produces DNA photoproducts that are blocks to DNA replication by normal replicative polymerases. A specialized, damage-specific, distributive polymerase, Pol H or Pol h, that is the product of the hRad30A gene, is required for replication past these photoproducts. This polymerase is absent from XP variant (XP-V) cells that must employ other mechanisms to negotiate blocks to DNA replication. These mechanisms include the use of alternative polymerases or recombination between sister chromatids. Replication forks arrested by UV damage in virus transformed XP-V cells degrade into DNA double strand breaks that are sites for recombination, but in normal cells arrested forks may be protected from degradation by p53 protein. These breaks are sites for binding a protein complex, hMre11/hRad50/Nbs1, that colocalizes with H2AX and PCNA, and can be visualized as immunofluorescent foci. The protein complexes need phosphorylation to activate their DNA binding capacity. Incubation of UV irradiated XP-V cells with the irreversible kinase inhibitor wortmannin, however, increased the yield of Mre11 focus-positive cells. One interpretation of this observation is that two classes of kinases are involved after UV irradiation. One would be a wortmannin-resistant kinase that phosphorylates the Mre11 complex. The other would be a wortmannin-sensitive kinase that phosphorylates and activates the p53/large T in SV40 transformed XP-V cells. The sensitive class corresponds to the PI3-kinases of ATM, ATR, and DNA-PK, but the resistant class remains to be identified. Alternatively, the elevated yield of Mre11 foci positive cells following wortmannin treatment may reflect an overall perturbation to the signaling cascades regulated by wortmannin-sensitive PI3 related kinases. In this scenario, wortmannin could compromise damage inducible-signaling pathways that maintain the stability of stalled forks, resulting in a further destabilization of stalled forks that then degrade, with the formation of DNA double strand breaks.
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PMID:DNA replication arrest in XP variant cells after UV exposure is diverted into an Mre11-dependent recombination pathway by the kinase inhibitor wortmannin. 1245 48

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.
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PMID:DNA double-strand breaks and gamma-H2AX signaling in the testis. 1253 28

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

We report cytologic and genetic data indicating that telomere dysfunction induces a DNA damage response in mammalian cells. Dysfunctional, uncapped telomeres, created through inhibition of TRF2, became associated with DNA damage response factors, such as 53BP1, gamma-H2AX, Rad17, ATM, and Mre11. We refer to the domain of telomere-associated DNA damage factors as a Telomere Dysfunction-Induced Focus (TIF). The accumulation of 53BP1 on uncapped telomeres was reduced in the presence of the PI3 kinase inhibitors caffeine and wortmannin, which affect ATM, ATR, and DNA-PK. By contrast, Mre11 TIFs were resistant to caffeine, consistent with previous findings on the Mre11 response to ionizing radiation. A-T cells had a diminished 53BP1 TIF response, indicating that the ATM kinase is a major transducer of this pathway. However, in the absence of ATM, TRF2 inhibition still induced TIFs and senescence, pointing to a second ATM-independent pathway. We conclude that the cellular response to telomere dysfunction is governed by proteins that also control the DNA damage response. TIFs represent a new tool for evaluating telomere status in normal and malignant cells suspected of harboring dysfunctional telomeres. Furthermore, induction of TIFs through TRF2 inhibition provides an opportunity to study the DNA damage response within the context of well-defined, physically marked lesions.
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PMID:DNA damage foci at dysfunctional telomeres. 1295 59

The non-homologous end joining pathway uses pre-existing proteins to repair DNA double-strand breaks induced by ionizing radiation. Here we describe manipulation of this pathway in living cells using a newly developed tool. We generated a single chain antibody variable fragment (scFv) that binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in the pathway. In contrast to existing pharmacologic inhibitors, the scFv binds a newly defined regulatory site outside the kinase catalytic domain. Although the scFv inhibits kinase activity only modestly, it completely blocks DNA end joining in a cell-free system. Microinjection of the scFv sensitizes human cells to radiation, as measured by a reduction in efficiency of colony formation and induction of apoptosis at an otherwise sublethal dose of 1.5 Gy. The scFv blocks non-homologous end joining in situ at a step subsequent to histone gamma-H2AX focus formation but preceding gamma-H2AX dephosphorylation. Blockage occurs in cells exposed to as little as 0.1 Gy, indicating that DNA-PKcs is essential for double-strand break repair even at low radiation doses. The ability to modify the radiation response in situ in living cells provides a link between biochemical, genetic and cytologic approaches to the study of double-strand break repair intermediates.
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PMID:Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase. 1453 Apr 33

Eukaryotic DNA is organized into nucleosomes and higher order chromatin structure, which plays an important role in the regulation of many nuclear processes including DNA repair. Non-homologous end-joining, the major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells, is mediated by a set of proteins including DNA-dependent protein kinase (DNA-PK). DNA-PK is comprised of a large catalytic subunit, DNA-PKcs, and its regulatory subunit, Ku. Current models predict that Ku binds to the ends of broken DNA and DNA-PKcs is recruited to form the active kinase complex. Here we show that DNA-PK can be activated by nucleosomes through the ability of Ku to bind to the ends of nucleosomal DNA, and that the activated DNA-PK is capable of phosphorylating H2AX within the nucleosomes. Histone acetylation has little effect on the steps of Ku binding to nucleosomes and subsequent activation of DNA-PKcs. However, acetylation largely enhances the phosphorylation of H2AX by DNA-PK, and this acetylation effect is observed when H2AX exists in the context of nucleosomes but not in a free form. These results suggest that the phosphorylation of H2AX, known to be important for DSB repair, can be regulated by acetylation and may provide a mechanistic basis on which to understand the recent observations that histone acetylation critically functions in repairing DNA DSBs.
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PMID:DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner. 1462 15


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