Rad17 recruits the MRE11‐RAD50‐NBS1 complex to regulate the cellular response to DNA double‐strand breaks

The MRE11‐RAD50‐NBS1 (MRN) complex is essential for the detection of DNA double‐strand breaks (DSBs) and initiation of DNA damage signaling. Here, we show that Rad17, a replication checkpoint protein, is required for the early recruitment of the MRN complex to the DSB site that is independent of MDC1 and contributes to ATM activation. Mechanistically, Rad17 is phosphorylated by ATM at a novel Thr622 site resulting in a direct interaction of Rad17 with NBS1, facilitating recruitment of the MRN complex and ATM to the DSB, thereby enhancing ATM signaling. Repetition of these events creates a positive feedback for Rad17‐dependent activation of MRN/ATM signaling which appears to be a requisite for the activation of MDC1‐dependent MRN complex recruitment. A point mutation of the Thr622 residue of Rad17 leads to a significant reduction in MRN/ATM signaling and homologous recombination repair, suggesting that Thr622 phosphorylation is important for regulation of the MRN/ATM signaling by Rad17. These findings suggest that Rad17 plays a critical role in the cellular response to DNA damage via regulation of the MRN/ATM pathway.     

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.     

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.     

Cytosolic DNA triggers mitochondrial apoptosis via DNA damage signaling proteins independently of AIM2 and RNA polymerase III

A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome.     

PIDD Death-Domain Phosphorylation by ATM Controls Prodeath versus Prosurvival PIDDosome Signaling

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of?signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.     

Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.     

Inactivation of ATM/ATR DNA Damage Checkpoint Promotes Androgen Induced Chromosomal Instability in Prostate Epithelial Cells

The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene have been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR) to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen-induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced autophosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis.     

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.     

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PK_cs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.     

Vaccinia-related kinase 1 (VRK1) is an upstream nucleosomal kinase required for the assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiation-induced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses.     

Rho GTPase independent regulation of ATM activation and cell survival by the RhoGEF Net1A

ATM activation following DNA damage is a critical event which is required for efficient DNA repair and cell survival, yet signaling mechanisms controlling its activation are incompletely understood. The RhoGEF Net1 has previously been reported to control Rho GTPase activation and downstream cell survival outcomes following double strand DNA damage. However the role of Net1 isoforms in controlling ATM-dependent cell signaling has not been assessed. In the present work we show that expression of the Net1A isoform is specifically required for efficient activation of ATM but not the related kinase DNA-PK after ionizing radiation. Surprisingly Net1A overexpression also potently suppresses ATM activation and phosphorylation of its substrate H2AX. This effect does not require catalytic activity towards RhoA or RhoB, and neither Rho GTPase affects ATM activation, on its own. Consistent with a role in controlling ATM activation, Net1A knockdown also impairs DNA repair and cell survival. Taken together these data indicate that Net1A plays a plays a previously unrecognized, Rho GTPase-independent role in controlling ATM activity and downstream signaling after DNA damage to impact cell survival.     

Activation of ataxia telangiectasia-mutated DNA damage checkpoint signal transduction elicited by herpes simplex virus infection

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection.     

RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.     

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain

Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.     

Regulation of Polo-like kinase 1 by DNA damage in mitosis. Inhibition of mitotic PLK-1 by protein phosphatase 2A

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. In addition to Plk1 functions in cell cycle, Plk1 is involved in DNA damage check-point in G2 phase. Normally, ataxia telangiectasia-mutated kinase (ATM) is a key enzyme involved in G2 phase cell cycle arrest following DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in a ATM/ATR-dependent manner. However, it is still unclear how Plk1 is regulated in response to DNA damage in mitosis in which Plk1 is already activated. Here, we show that treatment of mitotic cells with doxorubicin and gamma-irradiation inhibits Plk1 activity through dephosphorylation of Plk1, and cells were arrested in G2 phase. Treatments of the phosphatase inhibitors and siRNA experiments suggested that PP2A pathway might be involved in regulating mitotic Plk1 activity in mitotic DNA damage. Finally, we propose a novel pathway, which is connected between ATM/ATR/Chk and protein phosphatase-Plk1 in DNA damage response in mitosis.     

Functional interplay between the oxidative stress response and DNA damage checkpoint signaling for genome maintenance in aerobic organisms

The DNA damage checkpoint signaling pathway is a highly conserved surveillance mechanism that ensures genome integrity by sequential activation of protein kinase cascades. In mammals, the main pathway is orchestrated by two central sensor kinases, ATM and ATR, that are activated in response to DNA damage and DNA replication stress. Patients lacking functional ATM or ATR suffer from ataxia-telangiectasia (A-T) or Seckel syndrome, respectively, with pleiotropic degenerative phenotypes. In addition to DNA strand breaks, ATM and ATR also respond to oxidative DNA damage and reactive oxygen species (ROS), suggesting an unconventional function as regulators of intracellular redox status. Here, we summarize the multiple roles of ATM and ATR, and of their orthologs in Saccharomyces cerevisiae, Tel1 and Mec1, in DNA damage checkpoint signaling and the oxidative stress response, and discuss emerging ideas regarding the possible mechanisms underlying the elaborate crosstalk between those pathways. This review may provide new insights into the integrated cellular strategies responsible for maintaining genome stability in eukaryotes with a focus on the yeast model organism.     

ATM-mediated stabilization of hMutL DNA mismatch repair proteins augments p53 activation during DNA damage

Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. Here we show that DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response.