Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.     

Heat induces gammaH2AX foci formation in mammalian cells

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.     

Heat shock does not induce gammaH2AX foci formation but protects cells from N-methyl-N'-nitro-N-nitrosoguanidine-induced genotoxicity

The involvement of DNA damage in heat shock-induced cell death remains controversial. To investigate whether heat shock can induce DNA damage, we tested the induction of gammaH2AX foci formation, a sensitive indicator for DNA double strand breaks (DSBs), by heat shock treatment in several cell lines including HeLa, CHL, HepG2, and 293 cells, as well as human spermatozoa. Although heat shock treatment can decrease cell viability, no induction of gammaH2AX foci formation was observed in any of these cells. In addition, a p53-deficient cell line (U2OSE6tet24) and a flap endonuclease 1 (FEN1)-deficient cell line (FL-FEN1(-)) also did not show induction of gammaH2AX foci after heat shock treatment. Finally, it was found that 30min of pre-heat shock can inhibit gammaH2AX foci formation induced by an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which is known to induce gammaH2AX foci formation. On the other hand, heat shock after MNNG treatment did not affect the gammaH2AX foci formation induced by MNNG. Taken together, these data suggest that although heat shock might influence the gammaH2AX foci formation process, it does not induce DNA damage in the cells tested in this study.     

DNA double-strand breaks in human lymphocytes after single irradiation by low doses of pulsed X-rays: non-linear dose-response relationship

Effects of ionizing radiation registered in cells after low dose irradiation are still poorly understood. A pulsed mode of irradiation is even more problematic in terms of predicting the radiation-induced response in cells. Thus, the aim of this paper was to study and analyze the effects of dose and frequency of pulsed X-rays on the frequency of radiation-induced DNA double-strand breaks and their repair kinetics in human peripheral blood lymphocytes in vitro. Analysis of radiation-induced gammaH2AX and 53BP1 repair foci was used to assess the DNA damage in these cells. The dose-response curve of radiation-induced foci of both proteins has shown deviations from linearity to a higher effect in the 12-32 mGy dose range and a lower effect at 72 mGy. The dose-response curve was linear at doses higher than 100 mGy. The number of radiation-induced gammaH2AX and 53BP1 foci depended on the frequency of X-ray pulses: the highest effect was registered at 13 pulses per second. Moreover, slower repair kinetics was observed for those foci induced by very low doses with a nonlinear dose-response relationship.     

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.     

DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors

It has been reported that the phosphorylated form of histone variant H2AX (gammaH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using gammaH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce gammaH2AX foci in a 24h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce gammaH2AX foci formation in a time- and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of gammaH2AX foci in a time- and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce gammaH2AX foci formation in FL cells; however, AAF can induce gammaH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce gammaH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce gammaH2AX foci. Cell survival analyses indicated that the induction of gammaH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that gammaH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining gammaH2AX foci formation induced by a specific agent.     

Hypertonic saline enhances expression of phosphorylated histone H2AX after irradiation

Phosphorylation of histone H2AX at serine 139 occurs at sites surrounding DNA double-strand breaks, producing discrete spots called "foci" that are visible with a microscope after antibody staining. This modification is believed to create changes in chromatin structure and assemble various repair proteins at sites of DNA damage. To examine the role of chromatin structure, human SiHa cells were exposed to hypertonic salt solutions that are known to condense chromatin and sensitize cells to chromosome damage and killing by ionizing radiation. Postirradiation incubation in 0.5 M Na(+) increased gammaH2AX expression about fourfold as measured by flow cytometry and immunoblotting, and loss of gammaH2AX was inhibited in the presence of high salt. Focus size rather than the number of radiation-induced gammaH2AX foci was also increased about fourfold. When high-salt treatment was delayed for 1 h after irradiation, effects on focus size and retention were reduced. The increase in focus size was associated with a decrease in the rate of rejoining of double-strand breaks as measured using the neutral comet assay. We conclude that gammaH2AX expression after irradiation is sensitive to salt-induced changes in chromatin structure during focus formation, and that a large focus size may be an indication of a reduced ability to repair DNA damage.     

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.     

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.     

X-irradiation of cells on glass slides has a dose doubling impact

Immunofluorescence detection of gammaH2AX foci is a widely used tool to quantify the induction and repair of DNA double-strand breaks (DSBs) induced by ionising radiation. We observed that X-irradiation of mammalian cells exposed on glass slides induced twofold higher foci numbers compared to irradiation with gamma-rays. Here, we show that the excess gammaH2AX foci after X-irradiation are produced from secondary radiation particles generated from the irradiation of glass slides. Both 120 kV X-rays and (137)Cs gamma-rays induce approximately 20 gammaH2AX foci per Gy in cells growing on thin ( approximately 2 microm) plastic foils immersed in water. The same yield is obtained following gamma-irradiation of cells growing on glass slides. However, 120 kV X-rays produce approximately 40 gammaH2AX foci per Gy in cells growing on glass, twofold greater than obtained using cells irradiated on plastic surfaces. The same increase in gammaH2AX foci number is obtained if the plastic foil on which the cells are grown is irradiated on a glass slide. Thus, the physical proximity to the glass material and not morphological differences of cells growing on different surfaces accounts for the excess gammaH2AX foci. The increase in foci number depends on the energy and is considerably smaller for 25 kV relative to 120 kV X-rays, a finding which can be explained by known physical properties of radiation. The kinetics for the loss of foci, which is taken to represent the rate of DSB repair, as well as the Artemis dependent repair fraction, was similar following X- or gamma-irradiation, demonstrating that DSBs induced by this range of treatments are repaired in an identical manner.     

Spreading of mammalian DNA-damage response factors studied by ChIP-chip at damaged telomeres

Phosphorylated histone H2AX (gammaH2AX) is generated in nucleosomes flanking sites of DNA double-strand breaks, triggering the recruitment of DNA-damage response proteins such as MDC1 and 53BP1. Here, we study shortened telomeres in senescent human cells. We show that most telomeres trigger gammaH2AX formation, which spreads up to 570 kb into the subtelomeric regions. Furthermore, we reveal that the spreading patterns of 53BP1 and MDC1 are very similar to that of gammaH2AX, consistent with a structural link between these factors. Moreover, different subsets of telomeres signal in different cell lines, with those that signal tending to equate to the shortest telomeres of the corresponding cell line, thus linking telomere attrition with DNA-damage signalling. Notably, we find that, in some cases, gammaH2AX spreading is modulated in a manner suggesting that H2AX distribution or its ability to be phosphorylated is not uniform along the chromosome. Finally, we observe weak gammaH2AX signals at telomeres of proliferating cells, but not in hTERT immortalised cells, suggesting that low telomerase activity leads to telomere uncapping and senescence in proliferating primary cells.     

The double-edged sword of activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is required for Ig class switch recombination, a process that introduces DNA double-strand breaks in B cells. We show in this study that AID associates with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) promoting cell survival, presumably by resolving DNA double-strand breaks. Wild-type cells expressing AID mutants that fail to associate with DNA-PKcs or cells deficient in DNA-PKcs or 53BP1 expressing wild-type AID accumulate gammaH2AX foci, indicative of heightened DNA damage response. Thus, AID has two independent functions. AID catalyzes cytidine deamination that originates DNA double-strand breaks needed for recombination, and it promotes DNA damage response and cell survival. Our results thus resolve the paradox of how B cells undergoing DNA cytidine deamination and recombination exhibit heightened survival and suggest a mechanism for hyperIgM type II syndrome associated with AID mutants deficient in DNA-PKcs binding.     

Characterization of the role of a trimeric protein phosphatase complex in recovery from cisplatin-induced versus noncrosslinking DNA damage

Cisplatin (cis-diamminedichloroplatinum) and related chemotherapeutic DNA-crosslinking agents are widely used to treat human cancers. Saccharomyces cerevisiae with separate deletions of the genes encoding the trimeric protein serine/threonine phosphatase (Pph)3p-platinum sensitivity (Psy)4p-Psy2p complex, are more sensitive than the isogenic wild-type (WT) strain to cisplatin. We show here that cisplatin causes an enhanced intra-S-phase cell cycle delay in these three deletion mutants. The C-terminal tail of histone 2AX (H2AX) is hyperphosphorylated in the same mutants, and Pph3p is found to interact with phosphorylated H2AX (gammaH2AX). After cisplatin treatment is terminated, pph3Delta, psy4Delta and psy2Delta mutants are delayed as compared with the WT strain in the dephosphorylation of Rad53p. In contrast, only pph3Delta and psy2Delta cells are more sensitive than WT cells to methylmethanesulfonate, a noncrosslinking DNA-alkylating agent that is known to cause a Rad53p-dependent intra-S-phase cell cycle delay. Dephosphorylation of Rad53p and the recovery of chromosome replication are delayed in the same mutants, but not in psy4Delta cells. By comparison with their mammalian orthologues, the regulatory subunit Psy4p is likely to inhibit Pph3p catalytic activity. The presence of a weak but active Pph3p-Psy2p complex may allow psy4Delta cells to escape from the Rad53p-mediated cell cycle arrest. Overall, our data suggest that the trimeric Pph3p-Psy4p-Psy2p complex may dephosphorylate both gammaH2AX and Rad53p, the differences lying in the more stable interaction of the Pph3 phosphatase with gammaH2AX as opposed to a transient interaction with Rad53p.     

Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.     

Low-dose hyper-radiosensitivity is not caused by a failure to recognize DNA double-strand breaks

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.     

Activation of DNA damage response signaling in mouse embryonic stem cells

Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G1/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (γH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of γH2AX staining. Exposure of mESC to γ-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional γH2AX foci, which disappeared thereafter. To decrease the background of γH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G2/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for γH2AX. Irradiation induced the formation of γH2AX foci both in G2-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G1/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to γ-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing γH2AX foci formation, which are necessary for the activation of DNA damage response.     

Bloom syndrome cells undergo p53-dependent apoptosis and delayed assembly of BRCA1 and NBS1 repair complexes at stalled replication forks

Bloom syndrome (BS) is a hereditary disorder characterized by pre- and postnatal growth retardation, genomic instability, and cancer. BLM, the gene defective in BS, encodes a DNA helicase thought to participate in genomic maintenance. We show that BS human fibroblasts undergo extensive apoptosis after DNA damage specifically when DNA replication forks are stalled. Damage during S, but not G1, caused BLM to rapidly form foci with gammaH2AX at replication forks that develop DNA breaks. These BLM foci recruited BRCA1 and NBS1. Damaged BS cells formed BRCA1/NBS1 foci with markedly delayed kinetics. Helicase-defective BLM showed dominant-negative activity with respect to apoptosis, but not BRCA1/NBS1 recruitment, suggesting catalytic and structural roles for BLM. Strikingly, inactivation of p53 prevented the death of damaged BS cells and delayed recruitment of BRCA1/NBS1. These findings suggest that BLM is an early responder to damaged replication forks. Moreover, p53 eliminates cells that rapidly assemble BRCA1/NBS1 without BLM, suggesting that BLM is essential for timely BRCA1/NBS1 function.     

Oxidative stress induces H2AX phosphorylation in human spermatozoa

H2AX phosphorylation occurs following the induction of DNA double strand breaks (DSBs), thus collaborating with many other proteins to mediate important biological functions in somatic cells. In human spermatozoa, the present study showed that H(2)O(2) induced H2AX phosphorylation in a time- and dose-dependent manner. Moreover, such effect could be abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Meanwhile, the neutral comet assay also revealed DSBs production in correlation with H2AX phosphorylation assessed by flow cytometry. Besides H2AX phosphorylation, two other collaborating proteins, Rad50 and 53BP1, were also generated in spermatozoa after H(2)O(2) exposure. However, unlike in somatic FL cells, there were no distinctive focuses, but rather a whole nuclei staining pattern of these three proteins in spermatozoa. Additionally, gammaH2AX (the phosphorylated form of H2AX) staining in spermatozoa persisted despite the fact of a decrease in the number of gammaH2AX foci in FL cells after H(2)O(2) removal. Collectively, these results demonstrate that oxidative stress can induce H2AX phosphorylation in human spermatozoa through DSB induction, and that gammaH2AX may be used as a sensitive, novel marker for such DSBs. Moreover, the surveillance system involving gammaH2AX, Rad50, and 53BP1 in human spermatozoa cannot function effectively in DNA repair, but this system may possess other biological functions in response to DSBs.     

Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair

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.     

p53 suppression overwhelms DNA polymerase eta deficiency in determining the cellular UV DNA damage response

Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.