RESUMO
Histone H2AX becomes phosphorylated in chromatin domains flanking sites of DNA double-strand breakage associated with gamma-irradiation, meiotic recombination, DNA replication, and antigen receptor rearrangements. Here, we show that loss of a single H2AX allele compromises genomic integrity and enhances the susceptibility to cancer in the absence of p53. In comparison with heterozygotes, tumors arise earlier in the H2AX homozygous null background, and H2AX(-/-) p53(-/-) lymphomas harbor an increased frequency of clonal nonreciprocal translocations and amplifications. These include complex rearrangements that juxtapose the c-myc oncogene to antigen receptor loci. Restoration of the H2AX null allele with wild-type H2AX restores genomic stability and radiation resistance, but this effect is abolished by substitution of the conserved serine phosphorylation sites in H2AX with alanine or glutamic acid residues. Our results establish H2AX as genomic caretaker that requires the function of both gene alleles for optimal protection against tumorigenesis.
Assuntos
Aberrações Cromossômicas , DNA/metabolismo , Suscetibilidade a Doenças , Histonas/metabolismo , Animais , Linfócitos B/fisiologia , Células Cultivadas , DNA/genética , DNA/efeitos da radiação , Dano ao DNA , Reparo do DNA , Fibroblastos/citologia , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Raios gama , Histonas/genética , Cariotipagem , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Knockout , Fosforilação , Taxa de Sobrevida , Linfócitos T/citologia , Linfócitos T/fisiologia , Proteína Supressora de Tumor p53/genéticaAssuntos
Dano ao DNA , Reparo do DNA , Histonas/fisiologia , Animais , DNA/efeitos da radiação , HumanosRESUMO
Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.
Assuntos
Núcleo Celular/genética , Cromatina/genética , Dano ao DNA/genética , Células Eucarióticas/metabolismo , Histonas/deficiência , Animais , Linhagem Celular , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Fosforilação , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Western Blotting , Camptotecina/farmacologia , Enzimas Reparadoras do DNA , Inibidores Enzimáticos/farmacologia , Humanos , Hidrólise , Proteína Homóloga a MRE11 , Camundongos , Camundongos Knockout , Microscopia Confocal , Fosforilação , Inibidores da Topoisomerase I , Células Tumorais CultivadasRESUMO
Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX-/- mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.
Assuntos
Aberrações Cromossômicas , Reparo do DNA , Histonas/genética , Histonas/fisiologia , Recombinação Genética , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linfócitos B/fisiologia , Sequência de Bases , Ciclo Celular , Células Cultivadas , Senescência Celular , Dano ao DNA , Feminino , Marcação de Genes , Histonas/química , Switching de Imunoglobulina , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Contagem de Linfócitos , Masculino , Meiose , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Fosforilação , Espermatócitos/fisiologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (gamma-H2AX, also known as gamma-H2afx), which facilitate DNA double-strand break (DSB) repair, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and gamma-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (S mu)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.