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)

We used a proteomic approach to identify phosphopeptide-binding modules mediating signal transduction events in the DNA damage response pathway. Using a library of partially degenerate phosphopeptides, we identified tandem BRCT (BRCA1 carboxyl-terminal) domains in PTIP (Pax transactivation domain-interacting protein) and in BRCA1 as phosphoserine- or phosphothreonine-specific binding modules that recognize substrates phosphorylated by the kinases ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia- and RAD3-related) in response to gamma-irradiation. PTIP tandem BRCT domains are responsible for phosphorylation-dependent protein localization into 53BP1- and phospho-H2AX (gamma-H2AX)-containing nuclear foci, a marker of DNA damage. These findings provide a molecular basis for BRCT domain function in the DNA damage response and may help to explain why the BRCA1 BRCT domain mutation Met1775 --> Arg, which fails to bind phosphopeptides, predisposes women to breast and ovarian cancer.
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PMID:BRCT repeats as phosphopeptide-binding modules involved in protein targeting. 1457 10

Severe levels of hypoxia (oxygen concentrations of less that 0.02%) have been shown to induce a rapid S-phase arrest. The mechanism behind hypoxia-induced S-phase arrest is unclear, we show here that it was not mediated by a shortage of nucleosides and was not dependent on p53, p21 or Hif 1alpha status. The drugs aphidicolin and hydroxyurea both induce rapid replication arrest and have been used throughout the literature to study the ATR-mediated response to stalled replication. We have shown previously that hypoxia induces ATR-dependent phosphorylation of p53, Chk1 and histone H2AX. Using comet-assays to detect DNA-damage we found that both aphidicolin and hydroxyurea induced significant levels of DNA-damage while hypoxia did not. Here we show that like aphidicolin and hydroxyurea, hypoxia induces phosphorylation of Nbs1 at serine 343 and Rad17 serine 645. Hypoxia-dependent phosphorylation of Nbs1 and Rad17 was ATM-independent and therefore likely to be a result of the ATR kinase activity. In contrast, p53 was phosphorylated differentially in response to the three treatments considered here. p53 was phosphorylated at serine 15 in response to all three treatments but was only phosphorylated at serine 20 in response to the drug treatments. We propose that treatment with either aphidicolin or hydroxyurea leads to not only replication arrest but also DNA-damage and therefore both ATM and ATR-mediated signaling. In contrast replication arrest induced by severe hypoxia is sensed exclusively through ATR, with ATM only having a role to play after re-oxygenation.
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PMID:Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest. 1464 37

Mammalian TopBP1 is a BRCT domain-containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint. Here, we study its possible role during meiosis in mice. TopBP1 foci are abundant during early prophase I and localize mainly to histone gamma-H2AX-positive domains, where DNA double-strand breaks (required to initiate recombination) occur. Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint. In the synapsis-defective Fkbp6(-/-) mouse, TopBP1 heavily stains unsynapsed regions of chromosomes. We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue. Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint. Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint.
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PMID:TopBP1 and ATR colocalization at meiotic chromosomes: role of TopBP1/Cut5 in the meiotic recombination checkpoint. 1471 68

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.
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PMID:Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest. 1472 72

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

Disruption of the mechanisms that regulate cell-cycle checkpoints, DNA repair, and apoptosis results in genomic instability and the development of cancer in multicellular organisms. The protein kinases ATM and ATR, as well as their downstream substrates Chk1 and Chk2, are central players in checkpoint activation in response to DNA damage. Histone H2AX, ATRIP, as well as the BRCT-motif-containing molecules 53BP1, MDC1, and BRCA1 function as molecular adapters or mediators in the recruitment of ATM or ATR and their targets to sites of DNA damage. The increased chromosomal instability and tumor susceptibility apparent in mutant mice deficient in both p53 and either histone H2AX or proteins that contribute to the nonhomologous end-joining mechanism of DNA repair indicate that DNA damage checkpoints play a pivotal role in tumor suppression.
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PMID:DNA damage tumor suppressor genes and genomic instability. 1510 99

BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to become sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ATR (ataxia-telangiectasia-mutated/ATM and Rad3-related)-mediated phosphorylation of p53(Ser-15) following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53(Ser-15) efficiently. Phosphorylation of p53(Ser-15) is necessary for an IR-induced G(1)/S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, repressing p53(Ser-15) phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G(1)/S checkpoint arrest in response to IR but not UV radia-tion. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G(1)/S cell cycle progression after DNA damage.
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PMID:BRCA1-BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage. 1515 97

S(N)1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O(6)-methylguanine ((6Me)G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G(2) phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S(*)/T(*)Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage.
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PMID:Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase. 1517 64

Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX. Histone H2A is similarly regulated in Saccharomyces cerevisiae. The phosphorylated forms of H2AX and H2A, known as gamma-H2AX and gamma-H2A, are thought to be important for DNA repair, although their evolutionarily conserved roles are unknown. Here, we investigate gamma-H2A in the fission yeast Schizosaccharomyces pombe. We show that formation of gamma-H2A redundantly requires the ATR/ATM-related kinases Rad3 and Tel1. Mutation of the SQE motif to AQE (H2A-AQE) in the two histone H2A genes caused sensitivity to a wide range of genotoxic agents, increased spontaneous DNA damage, and impaired checkpoint maintenance. The H2A-AQE mutations displayed a striking synergistic interaction with rad22Delta (Rad52 homolog) in ionizing radiation (IR) survival. These phenotypes correlated with defective phosphorylation of the checkpoint proteins Crb2 and Chk1 and a failure to recruit large amounts of Crb2 to damaged DNA. Surprisingly, the H2A-AQE mutations substantially suppressed the IR hypersensitivity of crb2Delta cells by a mechanism that required the RecQ-like DNA helicase Rqh1. We propose that gamma-H2A modulates checkpoint and DNA repair through large-scale recruitment of Crb2 to damaged DNA. This function correlates with evidence that gamma-H2AX regulates recruitment of several BRCA1 carboxyl terminus domain-containing proteins (NBS1, 53BP1, MDC1/NFBD1, and BRCA1) in mammals.
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PMID:Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast. 1522 25

To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.
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PMID:Intra-S-phase checkpoint activation by direct CDK2 inhibition. 1522 29


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