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)

The histone H2A variant, H2AX, is a core component of chromatin that is phosphorylated in chromatin flanking DNA double strand breaks (DSBs). Here, we summarize H2AX functions and outline a specific "anchoring" model, that can explain the translocation prone phenotype of H2AX-deficient and H2AX/p53-deficient mice. We also discuss how this model of H2AX function could account for some aspects of the genomic instability and cancer prone human phenotypes associated with Ataxia Telangiectasia (AT), Nijmegen Breakage Syndrome (NBS), Ataxia Telangiectasia Like Disorder (ATLD), and Bloom's Syndrome (BS).
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PMID:H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity. 1471 78

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

Bloom's syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of gamma-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.
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PMID:Functional interaction between BLM helicase and 53BP1 in a Chk1-mediated pathway during S-phase arrest. 1536 58

The Bloom syndrome gene, BLM, encodes a RecQ DNA helicase that when absent from the cell results in genomic instability and cancer predisposition. We show here that BLM is a substrate for small ubiquitin-like modifier (SUMO) modification, with lysines at K317, K331, K334 and K347 being preferred sites of modification. Unlike normal BLM, a double mutant BLM protein with lysine to arginine substitutions at residues 317 and 331 was not modified by SUMO, and it failed to localize efficiently to the PML nuclear bodies. Rather, double mutant BLM protein induced the formation of DNA damage-induced foci (DDI) that contained BRCA1 protein and phosphorylated histone H2AX. Double mutant BLM only partially complemented the genomic instability phenotypes of Bloom syndrome cells as assessed by sister-chromatid exchange and micronuclei formation assays. These results constitute evidence that BLM is a DNA damage sensor that signals the formation of DDI, and they establish SUMO modification as a negative regulator of BLM's signaling function.
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PMID:Intra-nuclear trafficking of the BLM helicase to DNA damage-induced foci is regulated by SUMO modification. 1582 7

on-homologous end joining (NHEJ) and homologous recombination (HR) are pathways that repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, the repair of these breaks is influenced by histone acetylation. Therefore, we tested mammalian cells deleted for NHEJ (Ku80 or DNA Ligase IV) or altered for HR (breast cancer associated gene, Brca2, or Bloom's syndrome, Blm) for sensitivity to trichostatin A (TSA), a histone deacetylase inhibitor that is being investigated as an anti-cancer therapeutic. We show that cells mutated for Ku80 (ku80-/-) or DNA Ligase IV (lig 4-/-), but not cells mutated for Brca2 (brca2lex1/lex2) or Blm (blm(tm3Brd/tm4Brd)), are hypersensitive to TSA in a dose-dependent manner. TSA-induced toxicity stimulates apoptosis and cell cycle checkpoint responses independent of p53, but does not increase phosphorylated histone H2AX (-H2AX) as compared with a clastogenic agent, camptothecin, indicating that the quantity of DSBs is not the primary cause of TSA-induced cell death. In addition, we show that potential anti-cancer drugs (LY-294002 and vanillin) that inhibit the family of phosphatidylinositol 3 kinases that include the NHEJ protein, DNA-PKCS act in synergy with TSA to reduce the viability of HeLa cells in tissue culture presenting the possibility of using the two drugs in combination to treat cancer.
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PMID:Non-homologous end joining, but not homologous recombination, enables survival for cells exposed to a histone deacetylase inhibitor. 1617 81

Topoisomerase I-associated DNA single-strand breaks selectively trapped by camptothecins are lethal after being converted to double-strand breaks by replication fork collisions. BLM (Bloom's syndrome protein), a RecQ DNA helicase, and topoisomerase IIIalpha (Top3alpha) appear essential for the resolution of stalled replication forks (Holliday junctions). We investigated the involvement of BLM in the signaling response to Top1-mediated replication DNA damage. In BLM-complemented cells, BLM colocalized with promyelocytic leukemia protein (PML) nuclear bodies and Top3alpha. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (gamma-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3alpha, and PML protein and replication-dependent phosphorylation of BLM on threonine 99 (T99p-BLM). T99p-BLM was also observed following replication stress induced by hydroxyurea. Ataxia telangiectasia mutated (ATM) protein and AT- and Rad9-related protein kinases, but not DNA-dependent protein kinase, appeared to play a redundant role in phosphorylating BLM. Following camptothecin treatment, T99p-BLM colocalized with gamma-H2AX but not with Top3alpha or PML. Thus, BLM appears to dissociate from Top3alpha and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in gamma-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells.
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PMID:Phosphorylation of BLM, dissociation from topoisomerase IIIalpha, and colocalization with gamma-H2AX after topoisomerase I-induced replication damage. 1619 71

RecQ helicase BLM-deficient cells are characteristically hypersensitive to 4-nitroquinoline-1-oxide (4NQO). We recently reported that isogenic BLM-deficient cells (PNSG13) are more sensitive than BLM-complemented cells (PNSF5) to camptothecin, which specifically traps topoisomerase I cleavage complexes (Top1cc). We now report that PNSG13 are also 3.5-fold more sensitive to 4NQO compared with PNSF5 and that 4NQO induces higher levels of Top1cc and reduced histone gamma-H2AX in PSNG13 than in PNSF5. Similarly, 4NQO induces more Top1cc in primary fibroblasts from a patient with Bloom syndrome than in normal human fibroblasts. 4NQO also induces Top1cc in colon cancer HCT116 and HT29 cells in a time- and concentration-dependent fashion. Of note, distinct from camptothecin, the Top1cc produced by 4NQO accumulate progressively after 4NQO addition and persist following 4NQO removal. The Top1cc induced by 4NQO are detectable by alkaline elution. To examine the functional relevance of the Top1cc induced by 4NQO, we used two stable topoisomerase I small interfering RNA (siRNA) cell lines derived from HCT116 and MCF7 cells. Both topoisomerase I siRNA cell lines are resistant to 4NQO, indicating that Top1cc contribute to the cellular activity of 4NQO. Collectively, these data show that 4NQO is an effective inducer of cellular Top1cc. Because 4NQO does not directly trap Top1cc in biochemical assays, we propose that active metabolites of 4NQO trap Top1cc by forming DNA adducts. Induction of Top1cc and histone gamma-H2AX by 4NQO may contribute to the cellular effects of 4NQO, including its selective activity toward RecQ helicase BLM-deficient cells.
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PMID:4-nitroquinoline-1-oxide induces the formation of cellular topoisomerase I-DNA cleavage complexes. 1681 25

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.
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PMID:Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks. 1763 26

Five members of the RecQ subfamily of DEx-H-containing DNA helicases have been identified in both human and mouse, and mutations in BLM, WRN, and RECQ4 are associated with human diseases of premature aging, cancer, and chromosomal instability. Although a genetic disease has not been linked to RECQ1 mutations, RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage.(1) Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous gamma-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks.(2) Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin.(2) RECQ1 catalytic activities(3) and interactions with DNA repair proteins(2,4,5) are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis.
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PMID:Unique and important consequences of RECQ1 deficiency in mammalian cells. 1841 32

The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased gamma-H2AX foci. Because the increased gamma-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess gamma-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.
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PMID:SUMO modification regulates BLM and RAD51 interaction at damaged replication forks. 1995 65


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