ATMINistrating ATM signaling

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. We have recently identified an essential cofactor for ATM, ATMIN (for ATM INteractor). Several observations suggested that ATMIN plays a key role in ATM signalling. ATMIN and ATM protein stability were mutually dependent, which indicated an intimate physical and functional interaction. ATMIN bound ATM using a short carboxy-terminal motif, in a manner analogous to how another ATM cofactor, Nijmegen Breakage Syndrome protein 1 (NBS1), associates with ATM. ATMIN and NBS1 had complementary functions in ATM signalling. ATMIN was required for ATM signalling by chloroquine and hypotonic stress, but not after induction of double-stand breaks by ionizing radiation (IR), whereas NBS1 is required for ATM signalling by IR. This suggested competition of NBS1 and ATMIN for ATM binding in a signal-dependent fashion. Some implications of these findings for the ATM signalling pathway are discussed.     

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.     

Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage

Phosphorylation of NBS1, the product of the gene mutated in Nijmegen breakage syndrome (NBS), by ataxia telangiectasia mutated (ATM), the product of the gene mutated in ataxia telangiectasia, is required for activation of the S phase checkpoint in response to ionizing radiation (IR). However, NBS1 is also thought to play additional roles in the cellular response to DNA damage. To clarify these additional functions of NBS1, we generated NBS cell lines stably expressing various NBS1 mutants from retroviral vectors. The ATM-dependent activation of CHK2 by IR was defective in NBS cells but was restored by ectopic expression of wild-type NBS1. The defects in ATM-dependent activation of CHK2, S phase checkpoint control, IR-induced nuclear focus formation, and radiation sensitivity apparent in NBS cells were not corrected by expression of NBS1 mutants that lack an intact MRE11 binding domain, suggesting that formation of the NBS1-MRE11-RAD50 complex is required for the corresponding normal phenotypes. Expression of NBS1 proteins with mutated ATM-targeted phosphorylation sites (serines 278 or 343) did not restore S phase checkpoint control but did restore the ability of IR to activate CHK2 and to induce nuclear focus formation and normalized the radiation sensitivity of NBS cells. Expression of NBS1 containing mutations in the forkhead-associated or BRCA1 COOH terminus domains did not correct the defects in radiation sensitivity or nuclear focus formation but did restore S phase checkpoint control in NBS cells. Together, these data demonstrate that multiple functional domains of NBS1 are required for ATM-dependent activation of CHK2, nuclear focus formation, S phase checkpoint control, and cell survival after exposure to IR.     

Nbs1 is required for ATR-dependent phosphorylation events

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.     

ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.     

An essential function for NBS1 in the prevention of ataxia and cerebellar defects

Nijmegen breakage syndrome (NBS), ataxia telangiectasia and ataxia telangiectasia-like disorder (ATLD) show overlapping phenotypes such as growth retardation, microcephaly, cerebellar developmental defects and ataxia. However, the molecular pathogenesis of these neurological defects remains elusive. Here we show that inactivation of the Nbn gene (also known as Nbs1) in mouse neural tissues results in a combination of the neurological anomalies characteristic of NBS, ataxia telangiectasia and ATLD, including microcephaly, growth retardation, cerebellar defects and ataxia. Loss of Nbn causes proliferation arrest of granule cell progenitors and apoptosis of postmitotic neurons in the cerebellum. Furthermore, Nbn-deficient neuroprogenitors show proliferation defects (but not increased apoptosis) and contain more chromosomal breaks, which are accompanied by ataxia telangiectasia mutated protein (ATM)-mediated p53 activation. Notably, depletion of p53 substantially rescues the neurological defects of Nbn mutant mice. This study gives insight into the physiological function of NBS1 (the Nbn gene product) and the function of the DNA damage response in the neurological anomalies of NBS, ataxia telangiectasia and ATLD.     

Chk2 Activation Dependence on Nbs1 after DNA Damage

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.     

DNA Repair Cofactors ATMIN and NBS1 Are Required to Suppress T Cell Activation

Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1.     

Residual ataxia telangiectasia mutated protein function in cells from ataxia telangiectasia patients, with 5762ins137 and 7271T-->G mutations, showing a less severe phenotype

We have assessed several ataxia Telangiectasia mutated (ATM)-dependent functions in cells derived from ataxia telangiectasia patients, carrying either an ATM 5762ins137 splice site or a 7271T-->G missense mutation, with a less severe phenotype compared with the classical disorder. ATM kinase in vitro, from 5762ins137 cells, showed the same specific activity as ATM in normal cells, but the protein was present at low levels. In contrast, mutant ATM kinase activity in the 7271T-->G cells was only about 6% that of the activity in normal cells, although the level of mutant protein expressed was similar to normal cells. Phosphorylation of the DNA double strand break repair proteins Nbs1 and hMre11, following DNA damage, was observed in normal and 7271T-->G cells but was almost absent in both 5762ins137 and classical ataxia telangiectasia cells. The kinetics of p53 response was intermediate between normal and classical ataxia telangiectasia cells in both the 7271T-->G and 5762ins137 cells, but interestingly, c-Jun kinase activation following DNA damage was equally deficient in cell lines derived from all the ataxia telangiectasia patients. Our results indicate that levels of ATM kinase activity, but not induction of p53 or c-Jun kinase activity, in these cells correlate with the degree of neurological disorder in the patients.     

Radiation, DNA damage and cancer

The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasia and Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk of developing cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders provide valuable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role in sporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (ATM) has recently been shown to be a tumour suppressor gene in T-cell prolymphocytic leukaemia, and there is increasing evidence that individuals with one mutated ATM or Nijmegen breakage syndrome (NBS1) allele have an increased predisposition to cancer.     

Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses

Rad50/Mre11/NBS1 (R/M/N) is a multi-functional protein complex involved in DNA repair, cell cycle checkpoint activation, DNA replication and replication block-induced responses. Ionizing radiation (IR) induces the phosphorylation of NBS1 and nuclear foci formation of the complex. Although it has been suggested that the R/M/N complex is associated with DNA damage sites, we present here biochemical evidence for chromatin association of the complex. We show that the chromatin association of R/M/N is independent of IR and ataxia telangiectasia mutated (ATM). We also demonstrate that optimal chromatin association of the Rad50/Mre11/NBS1 proteins requires both the conserved forkhead-associated (FHA) and breast cancer C-terminus (BRCT) domains of NBS1. Moreover, both these domains of NBS1 are required for its phosphorylation on Ser343 but not on Ser278. Importantly, both the FHA and BRCT domains are essential for IR-induced foci (IRIF) formation of R/M/N and S phase checkpoint activation, but only the BRCT domain is needed for cell survival after IR. These data demonstrate that the FHA and BRCT domains of NBS1 are crucial for the functions of the R/M/N complex.     

Oxidative stress in ataxia telangiectasia

Ataxia telangiectasia is one of a group of recessive hereditary genomic instability disorders and is characterized by progressive neurodegeneration, immunodeficiency and cancer susceptibility. Heterozygotes for the mutated gene are more susceptible to cancer and to ischaemic heart disease. The affected gene, ATM (ataxia telangiectasia mutated), has been cloned and codes for a protein kinase (ATM), which orchestrates the cellular response to DNA double-strand breaks after ionising radiation. An underlying feature of ataxia telangiectasia is oxidative stress and there is chronic activation of stress response pathways in tissues showing pathology such as the cerebellum, but not in the cerebrum or liver. ATM has also been shown to be activated by insulin and to have a wider role in signal transduction and cell growth. Many, but not all, aspects of the phenotype can be attributed to a defective DNA damage response. The oxidative stress may result directly from accumulated DNA damage in affected tissues or ATM may have an additional role in sensing/modulating redox homeostasis. The basis for the observed tissue specificity of the oxidative damage in ataxia telangiectasia is not clear.     

A murine model of Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.     

TAO kinases mediate activation of p38 in response to DNA damage

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM.     

ATM activation by ionizing radiation requires BRCA1-associated BAAT1

ATM (ataxia telangiectasia mutated) is required for the early response to DNA-damaging agents such as ionizing radiation (IR) that induce DNA double-strand breaks. Cells deficient in ATM are extremely sensitive to IR. It has been shown that IR induces immediate phosphorylation of ATM at Ser(1981), leading to catalytic activation of the protein. We recently isolated a novel BRCA1-associated protein, BAAT1 (BRCA1-associated protein required for ATM activation-1), by yeast two-hybrid screening and found that BAAT1 also binds to ATM, localizes to double-strand breaks, and is required for Ser(1981) phosphorylation of ATM. Small interfering RNA-mediated stable or transient reduction of BAAT1 resulted in decreased phosphorylation of both ATM at Ser(1981) and CHK2 at Thr(68). Treatment of BAAT1-depleted cells with okadaic acid greatly restored phosphorylation of ATM at Ser(1981), suggesting that BAAT1 is involved in the regulation of ATM phosphatase. Protein phosphatase 2A-mediated dephosphorylation of ATM was partially blocked by purified BAAT1 in vitro. Significantly, acute loss of BAAT1 resulted in increased p53, leading to apoptosis. These results demonstrate that DNA damage-induced ATM activation requires a coordinated assembly of BRCA1, BAAT1, and ATM.     

Interaction of FANCD2 and NBS1 in the DNA damage response

Fanconi anaemia (FA) and Nijmegen breakage syndrome (NBS) are autosomal recessive chromosome instability syndromes with distinct clinical phenotypes. Cells from individuals affected with FA are hypersensitive to mitomycin C (MMC), and cells from those with NBS are hypersensitive to ionizing radiation. Here we report that both NBS cell lines and individuals with NBS are hypersensitive to MMC, indicating that there may be functional linkage between FA and NBS. In wild-type cells, MMC activates the colocalization of the FA subtype D2 protein (FANCD2) and NBS1 protein in subnuclear foci. Ionizing radiation activates the ataxia telangiectasia kinase (ATM)-dependent and NBS1-dependent phosphorylation of FANCD2, resulting in an S-phase checkpoint. NBS1 and FANCD2 therefore cooperate in two distinct cellular functions, one involved in the DNA crosslink response and one involved in the S-phase checkpoint response.     

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.