Duane R. Pilch

DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as γ, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. γ-Components, which appeared to be the only major novel components detected by mass or 32PO4incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. γ-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes γ-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 × 109 base pairs of a mammalian G1 genome, leads to the γ-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 × 106 base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, γ-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.

Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks

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 (γ-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 γ-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.

Histone H2A variants H2AX and H2AZ

Two of the nucleosomal histone families, H3 and H2A, have highly conserved variants with specialized functions. Recent studies have begun to elucidate the roles of two of the H2A variants, H2AX and H2AZ. H2AX is phosphorylated on a serine four residues from the carboxyl terminus in response to the introduction of DNA double-strand breaks, whether these breaks are a result of environmental insult, metabolic mistake, or programmed process. H2AZ appears to alter nucleosome stability, is partially redundant with nucleosome remodeling complexes, and is involved in transcriptional control.

AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching

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 (γ-H2AX, also known as γ-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 γ-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (Sµ)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.

H2AX Haploinsufficiency Modifies Genomic Stability and Tumor Susceptibility

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.

Distribution and Dynamics of Chromatin Modification Induced by a Defined DNA Double-Strand Break

BACKGROUND In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined. RESULTS We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein. gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11. gamma-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break. CONCLUSIONS Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.

Histone H2AX in DNA damage and repair.

No abstract available.

Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint‐blind DNA damage

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.

Techniques for γ-H2AX detection

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.

Genetic Analysis of Saccharomyces cerevisiae H2A Serine 129 Mutant Suggests a Functional Relationship Between H2A and the Sister-Chromatid Cohesion Partners Csm3–Tof1 for the Repair of Topoisomerase I-Induced DNA Damage

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.