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Summary The underlying relation between Parkinson’s disease (PD) etiopathology and its major risk factor, aging, is largely unknown. In light of the causative link between genome stability and aging, we investigate a possible nexus between DNA damage accumulation, aging, and PD by assessing aging-related DNA repair pathways in laboratory animal models and humans. We demonstrate that dermal fibroblasts from PD patients display flawed nucleotide excision repair (NER) capacity and that Ercc1 mutant mice with mildly compromised NER exhibit typical PD-like pathological alterations, including decreased striatal dopaminergic innervation, increased phospho-synuclein levels, and defects in mitochondrial respiration. Ercc1 mouse mutants are also more sensitive to the prototypical PD toxin MPTP, and their transcriptomic landscape shares important similarities with that of PD patients. Our results demonstrate that specific defects in DNA repair impact the dopaminergic system and are associated with human PD pathology and might therefore constitute an age-related risk factor for PD., Graphical Abstract, Highlights • Ercc1-mediated DNA repair is necessary for preservation of dopaminergic neurons • Mouse mutants with mild Ercc1 defects display signs of dopaminergic pathology • Mild Ercc1 dysfunction is sensitized to the prototypical PD neurotoxin MPTP • PD patients’ peripheral cells exhibit inefficient nucleotide excision repair, Sepe et al. demonstrate that the nucleotide excision repair (NER) gene Ercc1 is essential for dopaminergic neuronal integrity. Mild Ercc1 dysfunction is sufficient to elicit Parkinson’s disease (PD)-related dopaminergic pathology and to sensitize to PD-associated toxins. Furthermore, they show that PD patients’ peripheral cells are characterized by inefficient NER.

Using a zinc finger nuclease (ZFN) that creates a site-specific DNA double-strand break (DSB) at the endogenous ADH1 locus, we provide genetic evidence that histone H2AX and DSB-induced small RNAs modulate DNA repair by both non-homologous end-joining (NHEJ) and homologous recombination (HR) in the model plant Arabidopsis thaliana.

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.

A role for histone H2AX, one of the variants of the nucleosome core histone H2A, has been demonstrated in DNA repair, tumor suppression, apoptosis, and cell cycle checkpoint function. However, the physiological function and post-translational modification of histone H2AX during vertebrate development have not been elucidated. Here, we provide evidence showing that Xenopus histone H2AX (XH2AX) has a role in the anterior neural plate for eye field formation during Xenopus embryogenesis. A loss-of-function study clearly demonstrated a critical role of XH2AX in anterior neural specification. Through a differentiation assay with Xenopus animal cap embryonic stem cells, we confirmed that XH2AX is required for the activin-induced anterior neural specification of the ectoderm. Furthermore, we found that Chk1 is an essential kinase to phosphorylate histone XH2AX at Thr16, which is involved in the biological function of this histone. Taken together, our findings reveal that XH2AX has a specific role in anterior neural formation of Xenopus, which is mediated through phosphorylation of XH2AX at Thr16 by Chk1.

Polycyclic aromatic hydrocarbons (PAHs), wide-spread mutagenic and carcinogenic environmental pollutants, are consistently exposed to sunlight in the environment. Our previous paper showed that benzo[ a ]pyrene (BaP) exposed to solar-simulated light (SSL) induced phosphorylation of histone H2AX (γ-H2AX) [T. Toyooka, G. Ohnuki, Y. Ibuki, Solar-simulated light-exposed benzo[a]pyrene induces phosphorylation of histone H2AX, Mutat. Res. 650 (2008) 132–139], a marker of DNA double strand breaks. In this study, we found the ultraviolet B (UVB) region of SSL to produce photomodified BaP with high cytotoxicity and the ability to generate γ-H2AX. Degradation of BaP by SSL, resulting in an increase in cytotoxicity and the generation of γ-H2AX, was decreased by UVB-masking using a glass plate. Exposure to UVB itself increased the cytotoxicity of BaP and amount of γ-H2AX generated. Other PAHs, 1,2-benzoanthracene and 1,2:5,6-dibenzoanthracene, which absorb UVB, also showed enhanced cytotoxicity and the promoted the generation of γ-H2AX after exposure to SSL, whereas naphthalene and chrysene, which have low absorption in the UVB region, did not. These findings suggested that UVB is important for the degradation of PAHs having absorbance in this region, but that the production of genotoxic intermediates during the degradation process needs to be considered. UVB is a two-edged blade in environments, effectively degrading toxic chemicals but also producing genotoxic compounds as reactive intermediates.

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.

Benzene is a well known environmental carcinogen which causes myeloid leukemia. DNA damage induced by benzene metabolites such as hydroquinone (HQ) and p-benzoquinone (BQ) is one reason for the leukemogenesis. In this study, we showed that treatment with HQ and BQ quickly and clearly generated phosphorylated histone H2AX (gamma-H2AX) which has been recently considered an index of the production of double strand breaks (DSBs). HQ and BQ produced discrete foci of gamma-H2AX within the nucleus of HL-60 cells in a dose-dependent manner. gamma-H2AX appeared after the treatment with HQ and BQ for 2h, and increased time-dependently up to 4-8h. HQ and BQ increased intracellular oxidation, and an antioxidant, N-acetylcysteine, clearly inhibited the phosphorylation, suggesting that reactive oxygen species produced from HQ and BQ contributed to the generation. gamma-H2AX was sensitively detected after treatment with low concentrations of HQ and BQ, compared with the direct detection of DSBs by biased sinusoidal field gel electrophoresis and with the assessment of cytotoxicity based on cell survival. DSBs are the most serious form of DNA damage and are associated with genomic instability leading to myeloid leukemia. gamma-H2AX may be a useful tool for judging the genotoxicity of benzene metabolites sensitively.

H2AX is a histone variant which is present and ubiquitously distributed throughout the genome. An immunocytochemical assay using antibodies capable of recognizing histone H2AX phosphorylated at serine 139 (gammaH2AX) is very sensitive and is a specific indicator for the existence of a DNA double strand break. Although heat stress has been reported to induce the formation of gammaH2AX foci, no gammaH2AX foci formation was observed in several mammalian cell lines after heat shock. Since this was in contrast to earlier reports, the work described here was intended to verify that heat-induced gammaH2AX foci do form in mammalian cell lines other than the cell lines used in earlier reports concerning gammaH2AX foci formation. The cell lines used in this work includes cell lines with differing p53-gene status (H1299, H1299/neo, H1299/mp53 and H1299/wtp53 cells), various cancer cell lines (HeLa, HepG2, U2-OS cells), normal human cells (HEK-293 and AG1522), and cell lines established from other species (MEF normal mouse cells and CHL normal Chinese hamster cells). Exponentially growing cells were exposed to heat shock (42 degrees C for 6 h or 45.5 degrees C for 20 min) or to X-rays (3Gy). The presence of gammaH2AX was examined with immunocytochemistry and flow cytometry. Induction of gammaH2AX foci formation was observed in all of the mammalian cell lines used here after heat-treatment as well as after X-irradiation. However, the intensity of gammaH2AX was different in the different cell lines used. These results confirm that heat can induce gammaH2AX foci formation in many mammalian cell lines.

Histone H2AX undergoes phosphorylation on Ser 139 ({gamma}-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, {gamma}-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, {gamma}-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, {gamma}-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, {gamma}-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called gamma-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of gamma-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% gamma-H2AX-positive cells and in tissues fixed 1h after X-irradiation gamma-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of gamma-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the gamma-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of gamma-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.

Exposure of cells to hypertonic medium after X-irradiation results in a 3-4-fold increase in the phosphorylation of histone H2AX (gammaH2AX) at sites of radiation-induced DNA double-strand breaks. This increase was previously associated with salt-induced radiosensitization and inhibition of repair of DNA double-strand breaks. To examine possible mechanisms for the increase in foci size, chemical inhibitors of kinase and phosphatase activity and cell lines deficient in ATM and DNA-PK, two kinases known to phosphorylate H2AX, were examined. H2AX kinase and phosphatase activity were maintained in the presence of high salt. ATM mutant HT144 melanoma cells showed the expected 3-4-fold increase in H2AX phosphorylation in the presence of 0.5M Na(+). However, DNA-PKcs deficient M059J cells failed to respond to hypertonic treatment and M059J Fus1 cells corrected for this deficiency showed the expected increase in foci size. Although the active phosphoform of ATM, phosphoserine-1981, increased after irradiation, the level was unaffected by the addition of 0.5M Na(+). Instead, 0.5M Na(+) caused a partial redistribution of serine-1981-ATM to perinuclear regions. Hypertonic medium added after irradiation was effective in inhibiting rejoining of the radiation-induced double-strand breaks even in DNA-PK deficient M059J cells. We suggest that hypertonic treatment following irradiation inhibits double-strand break rejoining that in turn maintains DNA-PK activity at the site of the break, enhancing the size of the gammaH2AX foci.

Heat shock induces a variety of biological events including gene activation, cell cycle arrest, and apoptosis. Heat shock has recently been shown to be potentially useful when combined with radiation in cancer therapy, probably because, in mammalian cells, heat inhibits the repair of double-strand breaks (DSBs) induced by ionizing radiation. It remains unclear, however, whether heat shock by itself induces DSBs. In this communication, we present the first evidence that heat shock induces the phosphorylated form of histone H2AX, which is thought to be generated at the chromatin proximal to DSB sites. These results suggest that heat shock induces DSBs in mammalian cells and may provide direct evidence to explain previous reports on DSB-related events occurring after heat shock treatment.

In mammalian spermatogenesis, the X and Y chromosomes are transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation, MSCI), forming a condensed chromatin domain termed the sex or XY body [1–3]. The nucleosomal core histone H2AX is phosphorylated within the XY chromatin domain just prior to MSCI, and it has been hypothesized that this triggers the chromatin condensation and transcriptional repression [4, 5]. Here, we show that the kinase ATR localizes to XY chromatin at the onset of MSCI and that this localization is disrupted in mice with a mutant form of the tumor suppressor protein BRCA1. In the mutant pachytene cells, ATR is usually present at nonsex chromosomal sites, where it colocalizes with aberrant sites of H2AX phosphorylation; in these cells, there is MSCI failure. In rare pachytene cells, ATR does locate to XY chromatin, H2AX is then phosphorylated, a sex body forms, and MSCI ensues. These observations highlight an important role for BRCA1 in recruiting the kinase ATR to XY chromatin at the onset of MSCI and provide compelling evidence that it is ATR that phosphorylates H2AX and triggers MSCI.

Nijmegen breakage syndrome is a recessive genetic disorder, characterized by elevated sensitivity to ionizing radiation, chromosome instability and high frequency of malignancies. Since cellular features partly overlap with those of ataxia-telangiectasia (A-T), NBS was long considered an A-T clinical variant. NBS1, the product of the gene underlying the disease, contains three functional regions: the forkhead-associated (FHA) domain and BRCA1 C-terminus (BRCT) domain at the N-terminus, several SQ motifs (consensus phosphorylation sites by ATM and ATR kinases) at a central region and MRE11-binding region at the C-terminus. NBS1 forms a multimeric complex with hMRE11/hRAD50 nuclease at the C-terminus and recruits or retains them at the vicinity of sites of DNA damage by direct binding to histone H2AX, which is phosphorylated by ATM in response to DNA damage. The combination of the FHA/BRCT domains has a crucial role for the binding of NBS1 to H2AX. Thereafter, the NBS1 complex proceeds to rejoin double-strand breaks predominantly by homologous recombination repair in vertebrates, while it also might be involved in suppression of inter-chromosomal recombination even for V(D)J recombination. These processes collaborate with cell cycle checkpoints to facilitate DNA repair, while defects of these checkpoints in NBS cells are partial in nature. A possible explanation for these moderate defects are the redundancy of multiple checkpoint regulations in vertebrates, or the modulator role of NBS1, in which NBS1 amplifies ATM activation by accumulation of the MRN complex at damaged sites. This molecular link of NBS1 to ATM may explain the phenotypic similarity of NBS to A-T.

DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1(-/-)) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1(+/+)) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1(-/-) embryonic stem cell (ES) clones was also higher than that in Parp-1(+/+) ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and (32)P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB.

We employed gene targeting to study H2AX, a histone variant phosphorylated in chromatin surrounding DNA double-strand breaks. Mice deficient for both H2AX and p53 (H Δ/Δ P −/− ) rapidly developed immature T and B lymphomas and solid tumors. Moreover, H2AX haploinsufficiency caused genomic instability in normal cells and, on a p53-deficient background, early onset of various tumors including more mature B lymphomas. Most H2AX Δ/Δ p53 −/− or H2AX +/Δ p53 −/− B lineage lymphomas harbored chromosome 12 (IgH)/15 (c- myc ) translocations with hallmarks of either aberrant V(D)J or class switch recombination. In contrast, H2AX Δ/Δ p53 −/− thymic lymphomas had clonal translocations that did not involve antigen receptor loci and which likely occurred during cellular expansion. Thus, H2AX helps prevent aberrant repair of both programmed and general DNA breakage and, thereby, functions as a dosage-dependent suppressor of genomic instability and tumors in mice. Notably, H2AX maps to a cytogenetic region frequently altered in human cancers, possibily implicating similar functions in man.

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.

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 gamma, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. gamma-Components, which appeared to be the only major novel components detected by mass or 32PO4 incorporation 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. gamma-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 gamma-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 x 10(9) base pairs of a mammalian G1 genome, leads to the gamma-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 x 10(6) base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, gamma-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.

In a step toward clarifying how acute viral infections provoke the host DNA damage response, Tarakanova et al. (2007) characterized a gamma-herpesvirus protein, which phosphorylates histone H2AX during infection, suggesting that the virus actively initiates and benefits from the host DNA damage response.