Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia–telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.     

ATMIN defines an NBS1-independent pathway of ATM signalling

The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM co-localised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling.     

A novel ATM-dependent pathway regulates protein phosphatase 1 in response to DNA damage

Protein phosphatase 1 (PP1), a major protein phosphatase important for a variety of cellular responses, is activated in response to ionizing irradiation (IR)-induced DNA damage. Here, we report that IR induces the rapid dissociation of PP1 from its regulatory subunit inhibitor-2 (I-2) and that the process requires ataxia-telangiectasia mutated (ATM), a protein kinase central to DNA damage responses. In response to IR, ATM phosphorylates I-2 on serine 43, leading to the dissociation of the PP1-I-2 complex and the activation of PP1. Furthermore, ATM-mediated I-2 phosphorylation results in the inhibition of the Aurora-B kinase, the down-regulation of histone H3 serine 10 phosphorylation, and the activation of the G(2)/M checkpoint. Collectively, the results of these studies demonstrate a novel pathway that links ATM, PP1, and I-2 in the cellular response to DNA damage.     

Selective increase of dATP pools upon activation of deoxycytidine kinase in lymphocytes: implications in apoptosis

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction--presumably by supplying dATP or its analogues for the apoptosome formation--deserves consideration. Two-hour exposure of human tonsillar lymphocytes to 2-chloro-deoxyadenosine (CdA) led to a two-fold activation of dCK. This activation process was inhibited by pifithrin-alpha, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide-treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.     

Selective Increase of dATP Pools upon Activation of Deoxycytidine Kinase in Lymphocytes: Implications in Apoptosis

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction—presumably by supplying dATP or its analogues for the apoptosome formation—deserves consideration. Two‐hour exposure of human tonsillar lymphocytes to 2‐chloro‐deoxyadenosine (CdA) led to a two‐fold activation of dCK. This activation process was inhibited by pifithrin‐α, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide‐treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.     

Identification of Phosphorylation Sites on Human Deoxycytidine Kinase After Overexpression in Eucaryotic Cells

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [³²P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.     

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.     

Rapid activation of ATR by ionizing radiation requires ATM and Mre11

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.     

Genome-wide Reinforcement of Cohesin Binding at Pre-existing Cohesin Sites in Response to Ionizing Radiation in Human Cells

The cohesin complex plays a central role in genome maintenance by regulation of chromosome segregation in mitosis and DNA damage response (DDR) in other phases of the cell cycle. The ATM/ATR phosphorylates SMC1 and SMC3, two core components of the cohesin complex to regulate checkpoint signaling and DNA repair. In this report, we show that the genome-wide binding of SMC1 and SMC3 after ionizing radiation (IR) is enhanced by reinforcing pre-existing cohesin binding sites in human cancer cells. We demonstrate that ATM and SMC3 phosphorylation at Ser¹⁰⁸³ regulate this process. We also demonstrate that acetylation of SMC3 at Lys¹⁰⁵ and Lys¹⁰⁶ is induced by IR and this induction depends on the acetyltransferase ESCO1 as well as the ATM/ATR kinases. Consistently, both ESCO1 and SMC3 acetylation are required for intra-S phase checkpoint and cellular survival after IR. Although both IR-induced acetylation and phosphorylation of SMC3 are under the control of ATM/ATR, the two forms of modification are independent of each other and both are required to promote reinforcement of SMC3 binding to cohesin sites. Thus, SMC3 modifications is a mechanism for genome-wide reinforcement of cohesin binding in response to DNA damage response in human cells and enhanced cohesion is a downstream event of DDR.     

The HINT1 tumor suppressor regulates both gamma-H2AX and ATM in response to DNA damage

Hint1 is a haploinsufficient tumor suppressor gene and the underlying molecular mechanisms for its tumor suppressor function are unknown. In this study we demonstrate that HINT1 participates in ionizing radiation (IR)–induced DNA damage responses. In response to IR, HINT1 is recruited to IR-induced foci (IRIF) and associates with γ-H2AX and ATM. HINT1 deficiency does not affect the formation of γ-H2AX foci; however, it impairs the removal of γ-H2AX foci after DNA damage and this is associated with impaired acetylation of γ-H2AX. HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR. HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities. Our findings suggest that the tumor suppressor function of HINT1 is caused by, at least in part, its normal role in enhancing cellular responses to DNA damage by regulating the functions of both γ-H2AX and ATM.     

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G₁/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild-type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1^−/− murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.Key words: checkpoint control, DNA damage response, Nek1, ATM, ATR     

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.     

Deregulation of DNA damage signal transduction by herpesvirus latency-associated M2

Infected cells recognize viral replication as a DNA damage stress and elicit a DNA damage response that ultimately induces apoptosis as part of host immune surveillance. Here, we demonstrate a novel mechanism where the murine gamma herpesvirus 68 (gammaHV68) latency-associated, anti-interferon M2 protein inhibits DNA damage-induced apoptosis by interacting with the DDB1/COP9/cullin repair complex and the ATM DNA damage signal transducer. M2 expression constitutively induced DDB1 nuclear localization and ATM kinase activation in the absence of DNA damage. Activated ATM subsequently induced Chk activation and p53 phosphorylation and stabilization without eliciting H2AX phosphorylation and MRN recruitment to foci upon DNA damage. Consequently, M2 expression inhibited DNA repair, rendered cells resistant to DNA damage-induced apoptosis, and induced a G(1) cell cycle arrest. Our results suggest that gammaHV68 M2 blocks apoptosis-mediated intracellular innate immunity, which might ultimately contribute to its role in latent infection.     

Differentiation-induced radioresistance in muscle cells

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.     

Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage

Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.     

Skp2 E3 ligase integrates ATM activation and homologous recombination repair by ubiquitinating NBS1

The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.