DNA-PK is responsible for enhanced phosphorylation of histone H2AX under hypertonic conditions

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.     

Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors

In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.     

DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells

The phosphorylation of histone H2AX at serine 139 is one of the earliest responses of mammalian cells to ionizing radiation-induced DNA breaks. DNA breaks are also generated during the terminal stages of apoptosis when chromosomal DNA is cleaved into oligonucleosomal pieces. Apoptotic DNA fragmentation and the consequent chromatin condensation are important for efficient clearing of genomic DNA and nucleosomes and for protecting the organism from auto-immmunization and oncogenic transformation. In this study, we demonstrate that H2AX is phosphorylated during apoptotic DNA fragmentation in mouse, Chinese hamster ovary, and human cells. We have previously shown that ataxia telangiectasia mutated kinase (ATM) is primarily responsible for H2AX phosphorylation in murine cells in response to ionizing radiation. Interestingly, we find here that DNA-dependent protein kinase (DNA-PK) is solely responsible for H2AX phosphorylation during apoptosis while ATM is dispensable for the process. Moreover, the kinase activity of DNA-PKcs (catalytic subunit of DNA-PK) is specifically required for the induction of gammaH2AX. We further show that DNA-PKcs is robustly activated in apoptotic cells, as evidenced by autophosphorylation at serine 2056, before it is inactivated by cleavage. In contrast, ATM is degraded well before DNA fragmentation and gammaH2AX induction resulting in the predominance of DNA-PK during the later stages of apoptosis. Finally, we show that DNA-PKcs autophosphorylation and gammaH2AX induction occur only in apoptotic nuclei with characteristic chromatin condensation but not in non-apoptotic nuclei from the same culture establishing the most direct link between DNA fragmentation, DNA-PKcs activation, and H2AX phosphorylation. It is well established that DNA-PK is inactivated by cleavage late in apoptosis in order to forestall DNA repair. Our results demonstrate, for the first time, that DNA-PK is actually activated in late apoptotic cells and is able to initiate an early step in the DNA-damage response, namely H2AX phosphorylation, before it is inactivated by proteolysis.     

Tissue-specific DNA-PK-dependent H2AX phosphorylation and gamma-H2AX elimination after X-irradiation in vivo

Histone H2AX rapidly undergoes phosphorylation at Ser139 (γ-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of γ-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of γ-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of γ-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of γ-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of γ-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of γ-H2AX level is tissue-specific.     

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.     

Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis.

BACKGROUND: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. MATERIALS AND METHODS: HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. RESULTS: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. CONCLUSIONS: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation.     

Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes

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.     

ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke

Background  

In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX.     

Ionizing radiation-dependent gamma-H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (gamma-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of gamma-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, approximately 10% of IR-induced gamma-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, gamma-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.     

Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

Elevated level of oxygen (hyperoxia) is widely used in critical care units and in respiratory insufficiencies. In addition, hyperoxia has been implicated in many diseases such as bronchopulmonary dysplasia or acute respiratory distress syndrome. Although hyperoxia is known to cause DNA base modifications and strand breaks, the DNA damage response has not been adequately investigated. We have investigated the effect of hyperoxia on DNA damage signaling and show that hyperoxia is a unique stress that activates the ataxia telangiectasia mutant (ATM)- and Rad3-related protein kinase (ATR)-dependent p53 phosphorylations (Ser6, -15, -37, and -392), phosphorylation of histone H2AX (Ser139), and phosphorylation of checkpoint kinase 1 (Chk1). In addition, we show that phosphorylation of p53 (Ser6) and histone H2AX (Ser139) depend on both ATM and ATR. We demonstrate that ATR activation precedes ATM activation in hyperoxia. Finally, we show that ATR is required for ATM activation in hyperoxia. Taken together, we report that ATR is the major DNA damage signal transducer in hyperoxia that activates ATM.     

Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

Histone H2AX undergoes phosphorylation on Ser 139 (γ-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, γ-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, γ-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, γ-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, γ-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.     

DNA Double-Strand Break Formation upon UV-Induced Replication Stress Activates ATM and DNA-PKcs Kinases

The phosphatidylinositol 3-kinase-like protein kinases, including ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit), are the main kinases activated following various assaults on DNA. Although ATM and DNA-PKcs kinases are activated upon DNA double-strand breaks, evidence suggests that these kinases are rapidly phosphorylated by ATR kinase upon UV irradiation; thus, these kinases may also participate in the response to replication stress. Using UV-induced replication stress, we further characterize whether ATM and DNA-PKcs kinase activities are also involved in the cellular response. Contrary to the rapid activation of the ATR-dependent pathway, ATM-dependent Chk2 and KAP-1 phosphorylations, as well as DNA-PKcs Ser2056 autophosphorylation, reach their peak level at 4 to 8 h after UV irradiation. The delayed kinetics of ATM- and DNA-PKcs-dependent phosphorylations also correlated with a surge in H2AX phosphorylation, suggesting that double-strand break formation resulting from collapse of replication forks is responsible for the activation of ATM and DNA-PKcs kinases. In addition, we observed that some phosphorylation events initiated by ATR kinase in the response to UV were mediated by ATM at a later phase of the response. Furthermore, the S-phase checkpoint after UV irradiation was defective in ATM-deficient cells. These results suggest that the late increase of ATM activity is needed to complement the decreasing ATR activity for maintaining a vigilant checkpoint regulation upon replication stress.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and DNA-PK kinases that have distinct roles in different pathways of DSB repair. The γH2AX serves as a docking site for the accumulation of DNA repair proteins, and after rejoining of DSBs, it is released from chromatin. The molecular mechanism of γH2AX dephosphorylation is not clear. It is complicated and requires the activity of different proteins including phosphatases and chromatin-remodeling complexes. In this review, we summarize recently published data concerning the mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of the latest scientific research of the low-dose irradiation phenomenon are presented including the bystander effect and the adaptive response estimated by γH2AX detection in cells and tissues.     

Activation of ATM/ATR signaling in human embryonic stem cells after DNA damage

Embryonic stem cells (ESCs) are the progenitors of all adult cells; consequently, genomic abnormalities in them may be catastrophic for the developing organism. ESCs are characterized by high proliferation activity and do not stop in checkpoints upon DNA-damage executing only G₂/M delay after DNA damage. ATM and ATR kinases are key sensors of double-strand DNA breaks and activate downstream signaling pathways involving checkpoints, DNA repair, and apoptosis. We examined activation of ATM/ATR signaling in human ESCs and revealed that irradiation induced ATM, ATR, and Chk2 phosphorylation, and γH2AX foci formation and their colocalization with 53BP1 and Rad51 proteins. Interestingly, human ESCs exhibit noninduced γH2AX foci colocalized with Rad51 and marking single-strand DNA breaks. Next, we revealed the significant contribution of ATM, Chk1, and Chk2 kinases to G₂/M block after irradiation and ATM-dependent activation (phosphorylation) of p53 in human ESCs. However, p53 activation and subsequent induction of p21 ^Waf1 gene expression after DNA damage do not result in p21^Waf1 protein accumulation due to its proteasomal degradation.     

UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress

Ataxia-telangiectasia-mutated and Rad3-related (ATR) plays an essential role in the maintenance of genome integrity and cell viability. The kinase is activated in response to DNA damage and initiates a checkpoint signaling cascade by phosphorylating a number of downstream substrates including Chk1. Unlike ataxia-telangiectasia-mutated (ATM), which appears to be mainly activated by DNA double-strand breaks, ATR can be activated by a variety of DNA damaging agents. However, it is still unclear what triggers ATR activation in response to such diverse DNA lesions. One model proposes that ATR can directly recognize DNA lesions, while other recent data suggest that ATR is activated by a common single-stranded DNA (ssDNA) intermediate generated during DNA repair. In this study, we show that UV lesions do not directly activate ATR in vivo. In addition, ssDNA lesions created during the repair of UV damage are also not sufficient to activate the ATR-dependent pathway. ATR activation is only observed in replicating cells indicating that replication stress is required to trigger the ATR-mediated checkpoint cascade in response to UV irradiation. Interestingly, H2AX appears to be required for the accumulation of ATR at stalled replication forks. Together our data suggest that ssDNA at arrested replication forks recruits ATR and initiates ATR-mediated phosphorylation of H2AX and Chk1. Phosphorylated H2AX might further facilitate ATR activation by stabilizing ATR at the sites of arrested replication forks.     

Phosphorylation of histone H2AX at M phase in human cells without DNA damage response

A variant of histone H2A, H2AX, is phosphorylated on Ser139 in response to DNA double-strand breaks (DSBs), and clusters of the phosphorylated form of H2AX (gamma-H2AX) in nuclei of DSB-induced cells show foci at breakage sites. Here, we show phosphorylation of H2AX in a cell cycle-dependent manner without any detectable DNA damage response. Western blot and immunocytochemical analyses with the anti-gamma-H2AX antibody revealed that H2AX is phosphorylated at M phase in HeLa cells. In ataxia-telangiectasia cells lacking ATM kinase activity, gamma-H2AX was scarcely detectable in the mitotic chromosomes, suggesting involvement of ATM in M-phase phosphorylation of H2AX. Single-cell gel electrophoresis assay and Western blot analysis with the anti-phospho-p53 (Ser15) antibody indicated that H2AX in human M-phase cells is phosphorylated independently of DSB and DNA damage signaling. Even in the absence of DNA damage, phosphorylation of H2AX in normal cell cycle progression may contribute to maintenance of genomic integrity.