DNA damage signaling in response to double-strand breaks during mitosis

The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a “primary” DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11–Rad50–Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.     

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation

Activation of the melanocortin 1 receptor (MC1R) by α‐melanocortin (α‐MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)‐induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α‐MSH. α‐MSH up‐regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV‐induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3‐related (ATR) and ataxia telangiectasia mutated (ATM) and their respect‐ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation. These effects required functional MC1R and were absent in melanocytes expressing loss of function (LOF) MC1R. The levels of wild‐type p53‐induced phosphatase 1 (Wip1), which dephosphorylates γH2AX, correlated inversely with γH2AX. We propose that α‐MSH increases UV‐induced γH2AX to facilitate formation of DNA repair complexes and repair of DNA photoproducts, and LOF of MC1R compromises the DDR and genomic stability of melanocytes.     

Critical roles of tyrosyl‐DNA phosphodiesterases in cell tolerance to carnosol‐induced DNA damage

Carnosol is a natural compound with pharmacological action due to its anti‐cancer properties. However, the precise mechanism for its anti‐carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double‐strand breaks (DSBs). We also found that cells lacking tyrosyl‐DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl‐DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1‐DNA cleavage complex (TOP1cc) and TOP2‐DNA cleavage complex (TOP2cc). When comparing the accumulation of γ‐H2AX foci and the number of chromosomal aberrations (CAs) with wild‐type (WT) cells, the susceptivity of the TDP1^?/? and TDP2^?/? cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti‐cancer effect of carnosol.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

The involvement of human RECQL4 in DNA double‐strand break repair

Rothmund–Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas. The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double‐strand break (DSB) repair. The results show that RECQL4‐deficient fibroblasts are moderately sensitive to γ‐irradiation and accumulate more γH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB’s in the RECQL4‐deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy shows that RECQL4 is recruited early to laser‐induced DSBs and remains for a shorter duration than WRN and BLM, indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with γH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N‐terminus domain between amino acids 363–492, which shares no homology to recruitment domains of WRN and BLM to the DSBs. Further, the recruitment of RECQL4 to laser‐induced DNA damage is independent of functional WRN, BLM or ATM proteins. These results suggest distinct cellular dynamics for RECQL4 protein at the site of laser‐induced DSB and that it might play important roles in efficient repair of DSB’s.     

Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells

Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage.     

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)‐associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non‐tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H₂O₂). A population of HCEC survived H₂O₂‐induced oxidative stress via JNK‐dependent cell cycle arrests. Caspases, p21^WAF1 and γ‐H2AX were identified as JNK‐regulated proteins. Up‐regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan‐caspase inhibitor Z‐VAD‐FMK caused up‐regulation of γ‐H2AX, a DNA‐damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G₁‐phase as first response to oxidative stress and increased S‐phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non‐apoptotic function by promoting cells through G₁‐ and S‐phase by overriding the G₁/S‐ and intra‐S checkpoints despite DNA‐damage. This led to the accumulation of cells in the G₂/M‐phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ‐H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase‐dependent proteolytic degradation of the DNA‐damage checkpoint protein ATM that is upstream of γ‐H2AX. As a consequence, undetected DNA‐damage and increased proliferation were found in repeatedly H₂O₂‐exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non‐apoptotic function of caspases. Overexpression of upstream p‐JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.     

Mec1/ATR regulates the generation of single‐stranded DNA that attenuates Tel1/ATM signaling at DNA ends

Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double‐strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA‐coated single‐stranded DNA (ssDNA), which arises from 5′–3′ nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1‐ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53‐dependent and Rad53‐independent mechanisms. The mec1‐ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.     

DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.     

DNA damage response is suppressed by the high cyclin-dependent kinase 1 activity in mitotic mammalian cells

DNA damage response (DDR) is vital for genomic stability, and its deficiency is linked to tumorigenesis. Extensive studies in interphase (G(1)-S-G(2)) mammalian cells have revealed the mechanisms of DDR in great detail; however, how mitotic cells respond to DNA damage remains less defined. We report here that a full DDR is suppressed in mitotic mammalian cells until telophase/cytokinesis. Although early DDR markers such as the phosphorylations of ataxia telangiectasia mutated (ATM) and histone H2A.x (H2AX) can be readily detected, the ionizing radiation-induced foci (IRIF) formation of late DDR markers such as breast cancer type 1 susceptibility protein (BRCA1) and p53-binding protein 1 (53BP1) are absent until the telophase/cytokinesis stage. We further showed that the IR-induced ubiquitination cascade around DNA damage sites did not occur in mitotic cells, which explains, at least in part, why BRCA1 and 53BP1 cannot be recruited to the damaged sites. These observations indicate that DDR is suppressed in mitotic cells after the step of γH2AX formation. Not surprisingly, we found that the absence of a full DDR in mitotic cells was associated with the high cyclin-dependent kinase 1 (CDK1) activities. More 53BP1 IRIF could be detected when the irradiated mitotic cells were treated with a CDK1 inhibitor. Further, the activation of CDK5 in interphase cells impedes the formation of 53BP1 IRIF. Together, these results suggest that the DDR is suppressed by the high CDK1 activity in mitotic mammalian cells.     

γH2AX foci on apparently intact mitotic chromosomes: Not signatures of misrejoining events but signals of unresolved DNA damage

The presence of γH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between γH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of γH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the γH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal γH2AX foci, but very rarely are they signaled with Small γH2AX foci. To evaluate if Interstitial γH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial γH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a γH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all γH2AX foci—regardless their coincidence with a visible break—always colocalized with MRE11, but not with 53BP1. This pattern suggests that these γH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.     

Monoubiquitination of H2AX protein regulates DNA damage response signaling

Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR.     

Rotavirus activates a noncanonical ATM‐Chk2 branch of DNA damage response during infection to positively regulate viroplasm dynamics

Surveillance for maintaining genomic pristineness, a protective safeguard of great onco‐preventive significance, has been dedicated in eukaryotic cells to a highly conserved and synchronised signalling cascade called DNA damage response (DDR). Not surprisingly, foreign genetic elements like those of viruses are often potential targets of DDR. Viruses have evolved novel ways to subvert this genome vigilance by twisting canonical DDR to a skewed, noncanonical response through selective hijacking of some DDR components while antagonising the others. Though reported for many DNA and a few RNA viruses, potential implications of DDR have not been addressed yet in case of infection with rotavirus (RV), a double‐stranded RNA virus. In the present study, we aimed at the modulation of ataxia telangiectasia mutated (ATM)‐checkpoint kinase 2 (Chk2) branch of DDR in response to RV infection in vitro. We found activation of the transducer kinase ATM and its downstream effector Chk2 in RV‐SA11‐infected cells, the activation response being maximal at 6‐hr post infection. Moreover, ATM activation was found to be dependent on induction of the upstream sensor Mre11‐Rad50‐Nbs1 (MRN) complex. Interestingly, RV‐SA11‐mediated maximal induction of ATM‐Chk2 pathway was revealed to be neither preceded by occurrence of nuclear DNA damage nor transduced to formation of damage‐induced canonical nuclear foci. Subsequent investigations affirmed sequestration of MRN components as well as ATM‐Chk2 proteins away from nucleus into cytosolic RV replication factories (viroplasms). Chemical intervention targeting ATM and Chk2 significantly inhibited fusion and maturation of viroplasms leading to attenuated viral propagation. Cumulatively, the current study describes RV‐mediated activation of a noncanonical ATM‐Chk2 branch of DDR skewed in favour of facilitated viroplasm fusion and productive viral perpetuation.     

DNA damage response in renal ischemia–reperfusion and ATP-depletion injury of renal tubular cells

Renal ischemia–reperfusion leads to acute kidney injury (AKI) that is characterized pathologically by tubular damage and cell death, followed by tubular repair, atrophy and interstitial fibrosis. Recent work suggested the possible presence of DNA damage response (DDR) in AKI. However, the evidence is sketchy and the role and regulation of DDR in ischemic AKI remain elusive. In this study, we demonstrated the induction of phosphorylation of ATM, H2AX, Chk2 and p53 during renal ischemia–reperfusion in mice, suggesting DDR in kidney tissues. DDR was also induced in vitro during the recovery or “reperfusion” of renal proximal tubular cells (RPTCs) after ATP depletion. DDR in RPTCs was abrogated by supplying glucose to maintain ATP via glycolysis, indicating that the DDR depends on ATP depletion. The DDR was also suppressed by the general caspase inhibitor z-VAD and the overexpression of Bcl-2, supporting a role of apoptosis-associated DNA damage in the DDR. N-acetylcysteine (NAC), an antioxidant, suppressed the phosphorylation of ATM and p53 and, to a less extent, Chk2, but NAC increased the phosphorylation and nuclear foci formation of H2AX. Interestingly, NAC increased apoptosis, which may account for the observed H2AX activation. Ku55933, an ATM inhibitor, blocked ATM phosphorylation and ameliorated the phosphorylation of Chk2 and p53, but it increased H2AX phosphorylation and nuclear foci formation. Ku55933 also increased apoptosis in RPTCs following ATP depletion. The results suggest that DDR occurs during renal ischemia–reperfusion in vivo and ATP-depletion injury in vitro. The DDR is partially induced by apoptosis and oxidative stress-related DNA damage. ATM, as a sensor in the DDR, may play a cytoprotective role against tubular cell injury and death.