ABSTRACT
One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.
Subject(s)
Avian Proteins/physiology , DNA Repair/physiology , DNA-Binding Proteins/physiology , Histones/physiology , Rad51 Recombinase/metabolism , Recombination, Genetic , Animals , Avian Proteins/genetics , Avian Proteins/metabolism , Camptothecin/pharmacology , Cells, Cultured , Chickens , DNA Damage/physiology , DNA-Binding Proteins/genetics , Enzyme Inhibitors/pharmacology , Gamma Rays , Genomic Instability , Histones/genetics , Models, Genetic , Rad51 Recombinase/genetics , TransfectionABSTRACT
When a double-strand break (DSB) forms in DNA, many molecules of histone H2AX present in the chromatin flanking the break site are rapidly phosphorylated. The phosphorylated derivative of H2AX is named gamma-H2AX, and the phosphorylation site is a conserved serine four residues from the C-terminus, 139 in mammals and 129 in budding yeast. An antibody to gamma-H2AX reveals that the molecules form a gamma-focus at the DSB site. The gamma-focus increases in size rapidly for 10-30 min after formation, and remains until the break is repaired. Studies have revealed that small numbers of gamma-foci are present in cells even without the purposeful introduction of DNA DSBs. These cryptogenic foci increase in number during senescence in culture and aging in mice. This chapter presents techniques for revealing gamma-H2AX foci in cultured cells, in metaphase spreads from cultured cells, in tissues, and in yeast.
Subject(s)
Histones/analysis , Animals , Antibodies/immunology , Cells, Cultured , DNA Damage , Histones/immunology , Immunohistochemistry , In Situ Hybridization, Fluorescence , PhosphorylationABSTRACT
Collision between a topoisomerase I-DNA intermediate and an advancing replication fork represents a unique form of replicative damage. We have shown previously that yeast H2A serine 129 is involved in the recovery from this type of damage. We now report that efficient repair also requires proteins involved in chromatid cohesion: Csm3; Tof1; Mrc1, and Dcc1. Epistasis analysis defined several pathways involving these proteins. Csm3 and Tof1 function in a same pathway and downstream of H2A. In addition, the pathway involving H2A/Csm3/Tof1 is distinct from the pathways involving the Ctf8/Ctf18/Dcc1 complex, the Rad9 pathway, and another involving Mrc1. Our genetic studies suggest a role for H2A serine 129 in the establishment of specialized cohesion structure necessary for the normal repair of topoisomerase I-induced DNA damage.
Subject(s)
Chromatids/metabolism , DNA Damage , DNA Repair , DNA Topoisomerases, Type I/metabolism , Histones/genetics , Histones/metabolism , Mutation/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Rad52 DNA Repair and Recombination Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Serine/chemistry , Serine/geneticsABSTRACT
Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX Delta/Delta mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.
Subject(s)
DNA Damage , Histones/metabolism , Amino Acid Sequence , Animals , Chromatin/genetics , Chromatin/metabolism , Chromatin/radiation effects , Chromosomes/genetics , Chromosomes/metabolism , Chromosomes/radiation effects , DNA Repair , Histones/genetics , Humans , Mice , Molecular Sequence Data , Molecular Structure , Phosphorylation/radiation effects , Sequence Homology, Amino Acid , Time FactorsABSTRACT
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.
Subject(s)
Chromosome Aberrations , DNA/metabolism , Disease Susceptibility , Histones/metabolism , Animals , B-Lymphocytes/physiology , Cells, Cultured , DNA/genetics , DNA/radiation effects , DNA Damage , DNA Repair , Fibroblasts/cytology , Fibroblasts/physiology , Fibroblasts/radiation effects , Gamma Rays , Histones/genetics , Karyotyping , Lymphoma/genetics , Lymphoma/metabolism , Lymphoma/pathology , Mice , Mice, Knockout , Phosphorylation , Survival Rate , T-Lymphocytes/cytology , T-Lymphocytes/physiology , Tumor Suppressor Protein p53/geneticsABSTRACT
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.
Subject(s)
Cell Nucleus/genetics , Chromatin/genetics , DNA Damage/genetics , Eukaryotic Cells/metabolism , Histones/deficiency , Animals , Cell Line , DNA Damage/radiation effects , DNA Repair/genetics , DNA-Binding Proteins/genetics , Histones/genetics , Humans , Mice , Nuclear Proteins/genetics , Phosphorylation , Signal Transduction/geneticsABSTRACT
Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint control. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra-S-phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT-hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol-3-OH kinase (PI(3)K) motif at Ser 129 (h2a-s129a), the hypersensitivity was found to result from a failure to process full-length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for the H2A PI(3)K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra-S-phase checkpoint.
Subject(s)
DNA Damage/genetics , DNA Repair/physiology , Histones/chemistry , Histones/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Serine/metabolism , Animals , Camptothecin/pharmacology , Cell Cycle/physiology , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , Chromosomes, Fungal/radiation effects , DNA Damage/radiation effects , DNA Topoisomerases, Type I/metabolism , Electrophoresis, Gel, Pulsed-Field , Histones/genetics , Humans , Mutation, Missense/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/radiation effects , Serine/genetics , Topoisomerase I InhibitorsABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage , DNA Replication , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/metabolism , Histones/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Animals , Blotting, Western , Camptothecin/pharmacology , DNA Repair Enzymes , Enzyme Inhibitors/pharmacology , Humans , Hydrolysis , MRE11 Homologue Protein , Mice , Mice, Knockout , Microscopy, Confocal , Phosphorylation , Topoisomerase I Inhibitors , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Chromosome Aberrations , DNA Repair , Histones/genetics , Histones/physiology , Recombination, Genetic , Amino Acid Sequence , Animals , B-Lymphocytes/immunology , B-Lymphocytes/physiology , Base Sequence , Cell Cycle , Cells, Cultured , Cellular Senescence , DNA Damage , Female , Gene Targeting , Histones/chemistry , Immunoglobulin Class Switching , Infertility, Male/genetics , Infertility, Male/physiopathology , Lymphocyte Count , Male , Meiosis , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , Phosphorylation , Spermatocytes/physiology , T-Lymphocytes/immunology , T-Lymphocytes/physiologyABSTRACT
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.
