Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
 ...
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

DNA double-strand breaks represent the most potentially serious damage to a genome, and hence, many repair proteins are recruited to DNA damage sites by as yet poorly characterized sensor mechanisms. We clarified that NBS1 physically interacts with gamma-H2AX to form nuclear foci at DNA damage sites. The fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT) of NBS1 are essential for this physical interaction and focus formation of NBS1 in response to DNA damage. The inhibition of this interaction by introduction of anti-gamma-H2AX antibody into cells abolishes NBS1 foci formation in response to DNA damage. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for re-localization of the NBS1/hMRE11/hRAD50 complex to the vicinity of DNA damage. Moreover, the foci formation of DNA repair-related proteins containing BRCT domain, such as BRCA1, requires the interaction with gamma-H2AX in response to DNA damage. These findings indicate that the physical interaction between gamma-H2AX and DNA repair-related proteins is indispensable for the recruitment of these proteins. Further, it was recently reported that the NBS1/hMRE11/hRAD50 complex has a crucial role for both the recruitment of ATM to DNA damage sites and the subsequent activation of ATM. Therefore, both gamma-H2AX and the NBS1/hMRE11/hRAD50 complex might function for the initial recognition of DNA damage.
 ...
PMID:Molecular mechanism of the recruitment of NBS1/hMRE11/hRAD50 complex to DNA double-strand breaks: NBS1 binds to gamma-H2AX through FHA/BRCT domain. 1563 55

The Mre11/Rad50/NBS1 (MRN) complex is mutated in inherited genomic instability syndromes featuring cancer predisposition, mental retardation and immunodeficiency. It functions both in DNA double-strand break repair and in controlling the ataxia telangiectasia mutated (ATM) kinase during the response to these lesions. Patients inheriting homozygosity for an NBS1 hypomorphic allele display reduced phosphorylation of signaling factors such as Chk1, but not of chromatin-associated factor H2AX, after stresses that activate the ATM-related kinase, ATR. Therefore, we tested whether MRN has a global controlling role over the ATR kinase through the study of MRN deficiencies generated via RNA interference. We show for the first time that MRN is required for ATR-dependent phosphorylation of structural maintenance of chromosomes 1 (Smc1), which acts within chromatin to ensure sister chromatid cohesion and to effect several DNA damage responses. We have uncovered novel phenotypes caused by MRN deficiency that support a functional link between this complex, ATR and Smc1, including hypersensitivity to UV exposure, a defective UV responsive intra-S phase checkpoint and a specific pattern of genomic instability. In addition, certain ATR-dependent responses do not require MRN. These studies demonstrate that there is indeed a controlling role for MRN over the ATR kinase and have established that the downstream events under this control are broad, including both chromatin-associated and diffuse signaling factors, but may not be universal. These studies contribute to our understanding of the central role that MRN plays in damage detection and signaling, which serve to maintain genomic stability and resist neoplastic transformation.
 ...
PMID:Rad50 depletion impacts upon ATR-dependent DNA damage responses. 1608 84

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.
 ...
PMID:Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. 1661 11

Vorinostat (suberoylanilide hydroxamic acid) is the prototype of a family of hybrid polar compounds that can induce growth arrest in transformed cells and shows promise for the treatment of cancer. Vorinostat specifically binds to and inhibits the activity of histone deacetylases resulting in acetylation of nucleosomal histones and an activation of gene transcription. Because histone deacetylases modulate chromatin structure and gene expression, both of which can influence radioresponse, this study was designed to examine the capacity of Vorinostat to influence radiation response in human tumor cells and investigate the mechanism underlying these interactions. Vorinostat induced hyperacetylation of histone H4 in a dose-dependent manner. We tested its ability to radiosensitize three human tumor cell lines (A375, MeWo, and A549) using clonogenic cell survival assays. Clonogenic cell survival assay showed that Vorinostat significantly radiosensitized all three tumor cell lines, substantially reducing the surviving fraction at 2 Gy. We examined potential mechanisms that may contribute to the enhanced radiation response induced by Vorinostat. Vorinostat and radiation alone did not induce apoptosis in the melanoma cell line. However, enhanced apoptosis was observed when cells were exposed to both Vorinostat and radiation, suggesting that Vorinostat renders tumor cells more susceptible to radiation-induced apoptosis. Results from DNA damage repair analysis in cultured A375 cells showed that Vorinostat had a strong inhibitory effect on the nonhomologous end joining pathway after radiation. A detailed examination of the involvement of the DNA repair pathway following Vorinostat treatment showed that Vorinostat reduced the expression of the repair-related genes Ku70, Ku80, and Rad50 in A375 cells as detected by Western blot analysis. We also examined gamma-H2AX phosphorylation as a predictive marker of radiotherapy response to Vorinostat and observed that the combination of Vorinostat and radiation caused a prolongation of expression of DNA repair proteins such as gamma-H2AX. Overall, we conclude that Vorinostat enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation.
 ...
PMID:Vorinostat, a histone deacetylase inhibitor, enhances the response of human tumor cells to ionizing radiation through prolongation of gamma-H2AX foci. 1692 17

H2AX phosphorylation occurs following the induction of DNA double strand breaks (DSBs), thus collaborating with many other proteins to mediate important biological functions in somatic cells. In human spermatozoa, the present study showed that H(2)O(2) induced H2AX phosphorylation in a time- and dose-dependent manner. Moreover, such effect could be abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Meanwhile, the neutral comet assay also revealed DSBs production in correlation with H2AX phosphorylation assessed by flow cytometry. Besides H2AX phosphorylation, two other collaborating proteins, Rad50 and 53BP1, were also generated in spermatozoa after H(2)O(2) exposure. However, unlike in somatic FL cells, there were no distinctive focuses, but rather a whole nuclei staining pattern of these three proteins in spermatozoa. Additionally, gammaH2AX (the phosphorylated form of H2AX) staining in spermatozoa persisted despite the fact of a decrease in the number of gammaH2AX foci in FL cells after H(2)O(2) removal. Collectively, these results demonstrate that oxidative stress can induce H2AX phosphorylation in human spermatozoa through DSB induction, and that gammaH2AX may be used as a sensitive, novel marker for such DSBs. Moreover, the surveillance system involving gammaH2AX, Rad50, and 53BP1 in human spermatozoa cannot function effectively in DNA repair, but this system may possess other biological functions in response to DSBs.
 ...
PMID:Oxidative stress induces H2AX phosphorylation in human spermatozoa. 1706 97

The chemotherapeutic agent temozolomide produces O(6)-methylguanine (O6MG) in DNA, which triggers futile DNA mismatch repair, DNA double-strand breaks (DSB), G(2) arrest, and ultimately cell death. Because the protein complex consisting of Mre11/Rad50/Nbs1 (MRN complex) plays a key role in DNA damage detection and signaling, we asked if this complex also played a role in the cellular response to temozolomide. Temozolomide exposure triggered the assembly of MRN complex into chromatin-associated nuclear foci. MRN foci formed significantly earlier than gamma-H2AX and 53BP1 foci that assembled in response to temozolomide-induced DNA DSBs. MRN foci formation was suppressed in cells that incurred lower levels of temozolomide-induced O6MG lesions and/or had decreased mismatch repair capabilities, suggesting that the MRN foci formed not in response to temozolomide-induced DSB but rather in response to mismatch repair processing of mispaired temozolomide-induced O6MG lesions. Consistent with this idea, the MRN foci colocalized with those of proliferating cell nuclear antigen (a component of the mismatch repair complex), and the MRN complex component Nbs1 coimmunoprecipitated with the mismatch repair protein Mlh1 specifically in response to temozolomide treatment. Furthermore, small inhibitory RNA-mediated suppression of Mre11 levels decreased temozolomide-induced G(2) arrest and cytotoxicity in a manner comparable to that achieved by suppression of mismatch repair. These data show that temozolomide-induced O6MG lesions, acted upon by the mismatch repair system, drive formation of the MRN complex foci and the interaction of this complex with the mismatch repair machinery. The MRN complex in turn contributes to the control of temozolomide-induced G(2) arrest and cytotoxicity, and as such is an additional determining factor in glioma sensitivity to DNA methylating chemotherapeutic drugs such as temozolomide.
 ...
PMID:The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G2 arrest and cytotoxicity. 1712 22

Hydroxyurea reduces DNA replication by nucleotide deprivation, whereas UV damage generates DNA photoproducts that directly block replication fork progression. We show that the low fidelity class Y polymerase Pol eta is recruited to proliferating cell nuclear antigen at replication forks both by hydroxyurea and UV light. Under nucleotide deprivation, Pol eta allows cells to accumulate at the G1/S boundary by facilitating slow S-phase progression and promotes apoptosis. Normal cells consequently enter apoptosis at a faster rate than Pol eta-deficient cells. Coincident with hydroxyurea-induced S-phase delay, Pol eta-deficient cells undergo more replication fork breakage and accumulate more foci of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX. We conclude that under conditions of nucleotide deprivation, Pol eta is required for S-phase progression but is proapoptotic. However, as Pol eta is reported to require higher nucleotide concentrations than class B replicative polymerases, its recruitment by hydroxyurea requires it to function under suboptimal conditions. Our results suggest that hydroxyurea-induced apoptosis occurs at the G1/S boundary and that initiation of the S-phase requires greater nucleotide concentrations than does S-phase progression.
 ...
PMID:Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea. 1736 53

Commonly used antitumor agents, such as DNA topoisomerase I/II poisons, kill cancer cells by creating nonrepairable DNA double-strand breaks (DSBs). To repair DSBs, error-free homologous recombination (HR), and/or error-prone nonhomologous end joining (NHEJ) are activated. These processes involve the phosphatidylinositol 3'-kinase-related kinase family of serine/threonine enzymes: ataxia telangiectasia mutated (ATM), ATM- and Rad3-related for HR, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ. Alterations in these repair processes can cause drug/radiation resistance and increased genomic instability. beta-Lapachone (beta-lap; also known as ARQ 501), currently in phase II clinical trials for the treatment of pancreatic cancer, causes a novel caspase- and p53-independent cell death in cancer cells overexpressing NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 catalyzes a futile oxidoreduction of beta-lap leading to reactive oxygen species generation, DNA breaks, gamma-H2AX foci formation, and hyperactivation of poly(ADP-ribose) polymerase-1, which is required for cell death. Here, we report that beta-lap exposure results in NQO1-dependent activation of the MRE11-Rad50-Nbs-1 complex. In addition, ATM serine 1981, DNA-PKcs threonine 2609, and Chk1 serine 345 phosphorylation were noted; indicative of simultaneous HR and NHEJ activation. However, inhibition of NHEJ, but not HR, by genetic or chemical means potentiated beta-lap lethality. These studies give insight into the mechanism by which beta-lap radiosensitizes cancer cells and suggest that NHEJ is a potent target for enhancing the therapeutic efficacy of beta-lap alone or in combination with other agents in cancer cells that express elevated NQO1 levels.
 ...
PMID:Nonhomologous end joining is essential for cellular resistance to the novel antitumor agent, beta-lapachone. 1763 5

Cellular accumulation of DNA damage has been widely implicated in cellular senescence, aging, and premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is linked to accumulation of DNA double-strand breaks (DSBs), which results in genome instability. However, how DSBs accumulate in cells despite the presence of intact DNA repair proteins remains unknown. Here we report that the recruitment of DSB repair factors Rad50 and Rad51 to the DSB sites, as marked by gamma-H2AX, was impaired in human HGPS and Zmpste24-deficient cells. Consistently, the progeria-associated DSBs appeared to be unrepairable although DSBs induced by camptothecin were efficiently removed in the progeroid cells. We also found that these progeroid cells exhibited nuclear foci of xeroderma pigmentosum group A (XPA), a unique nucleotide excision repair protein. Strikingly, these XPA foci colocalized with the DSB sites in the progeroid cells. This XPA-DSB association was further confirmed and found to be mediated by DNA, using a modified chromatin immunoprecipitation assay and coimmunoprecipitation. RNA interference (RNAi) knockdown of XPA in HGPS cells partially restored DSB repair as evidenced by Western blot analysis, immunofluorescence and comet assays. We propose that the uncharacteristic localization of XPA to or near DSBs inhibits DSB repair, thereby contributing to the premature aging phenotypes observed in progeria arising from genetic defects in prelamin A maturation.
 ...
PMID:Involvement of xeroderma pigmentosum group A (XPA) in progeria arising from defective maturation of prelamin A. 1784 22


<< Previous 1 2 3 4 5 6 Next >>