A rat model of ataxia-telangiectasia: evidence for a neurodegenerative phenotype.

Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm knockout rat model that does not display cerebellar atrophy but instead paralysis and spinal cord atrophy, reminiscent of that seen in older patients and milder forms of the disorder. Loss of Atm in neurons and glia leads to accumulation of cytosolic DNA, increased cytokine production and constitutive activation of microglia consistent with a neuroinflammatory phenotype. Rats lacking ATM had significant loss of motor neurons and microgliosis in the spinal cord, consistent with onset of paralysis. Since short term treatment with steroids has been shown to improve the neurological signs in A-T patients we determined if that was also the case for Atm-deficient rats. Betamethasone treatment extended the lifespan of Atm knockout rats, prevented microglial activation and significantly decreased neuroinflammatory changes and motor neuron loss. These results point to unrepaired damage to DNA leading to significant levels of cytosolic DNA in Atm-deficient neurons and microglia and as a consequence activation of the cGAS-STING pathway and cytokine production. This in turn would increase the inflammatory microenvironment leading to dysfunction and death of neurons. Thus the rat model represents a suitable one for studying neurodegeneration in A-T and adds support for the use of anti-inflammatory drugs for the treatment of neurodegeneration in A-T patients.

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

RAD50 phosphorylation promotes ATR downstream signaling and DNA restart following replication stress.

The MRE11/RAD50/NBN (MRN) complex plays a key role in detecting DNA double-strand breaks, recruiting and activating ataxia-telangiectasia mutated and in processing the breaks. Members of this complex also act as adaptor molecules for downstream signaling to the cell cycle and other cellular processes. Somewhat more controversial are the results to support a role for MRN in the ataxia-telangiectasia and Rad3-related (ATR) activation and signaling. We provide evidence that RAD50 is required for ATR activation in mammalian cells in response to DNA replication stress. It is in turn phosphorylated at a specific site (S635) by ATR, which is required for ATR signaling through Chk1 and other downstream substrates. We find that RAD50 phosphorylation is essential for DNA replication restart by promoting loading of cohesin at these sites. We also demonstrate that replication stress-induced RAD50 phosphorylation is functionally significant for cell survival and cell cycle checkpoint activation. These results highlight the importance of the adaptor role for a member of the MRN complex in all aspects of the response to DNA replication stress.

Loss of caspase-2 augments lymphomagenesis and enhances genomic instability in Atm-deficient mice.

Caspase-2, the most evolutionarily conserved member of the caspase family, has been shown to be involved in apoptosis induced by various stimuli. Our recent work indicates that caspase-2 has putative functions in tumor suppression and protection against cellular stress. As such, the loss of caspase-2 enhances lymphomagenesis in Eµ-Myc transgenic mice, and caspase-2 KO (Casp2(-/-)) mice show characteristics of premature aging. However, the extent and specificity of caspase-2 function in tumor suppression is currently unclear. To further investigate this, ataxia telangiectasia mutated KO (Atm(-/-)) mice, which develop spontaneous thymic lymphomas, were used to generate Atm(-/-)Casp2(-/-) mice. Initial characterization revealed that caspase-2 deficiency enhanced growth retardation and caused synthetic perinatal lethality in Atm(-/-) mice. A comparison of tumor susceptibility demonstrated that Atm(-/-)Casp2(-/-) mice developed tumors with a dramatically increased incidence compared with Atm(-/-) mice. Atm(-/-)Casp2(-/-) tumor cells displayed an increased proliferative capacity and extensive aneuploidy that coincided with elevated oxidative damage. Furthermore, splenic and thymic T cells derived from premalignant Atm(-/-)Casp2(-/-) mice also showed increased levels of aneuploidy. These observations suggest that the tumor suppressor activity of caspase-2 is linked to its function in the maintenance of genomic stability and suppression of oxidative damage. Given that ATM and caspase-2 are important components of the DNA damage and antioxidant defense systems, which are essential for the maintenance of genomic stability, these proteins may synergistically function in tumor suppression by regulating these processes.

A patient-derived olfactory stem cell disease model for ataxia-telangiectasia.

The autosomal recessive disorder ataxia-telangiectasia (A-T) is characterized by genome instability, cancer predisposition and neurodegeneration. Although the role of ataxia-telangiectasia mutated (ATM) protein, the protein defective in this syndrome, is well described in the response to DNA damage, its role in protecting the nervous system is less clear. We describe the establishment and characterization of patient-specific stem cells that have the potential to address this shortcoming. Olfactory neurosphere (ONS)-derived cells were generated from A-T patients, which expressed stem cell markers and exhibited A-T molecular and cellular characteristics that included hypersensitivity to radiation, defective radiation-induced signaling and cell cycle checkpoint defects. Introduction of full-length ATM cDNA into these cells corrected defects in the A-T cellular phenotype. Gene expression profiling and pathway analysis revealed defects in multiple cell signaling pathways associated with ATM function, with cell cycle, cell death and DNA damage response pathways being the most significantly dysregulated. A-T ONS cells were also capable of differentiating into neural progenitors, but they were defective in neurite formation, number of neurites and length of these neurites. Thus, ONS cells are a patient-derived neural stem cell model that recapitulate the phenotype of A-T, do not require genetic reprogramming, have the capacity to differentiate into neurons and have potential to delineate the neurological defect in these patients.

ATM protein-dependent phosphorylation of Rad50 protein regulates DNA repair and cell cycle control.

The Mre11/Rad50/NBN complex plays a central role in coordinating the cellular response to DNA double-strand breaks. The importance of Rad50 in that response is evident from the recent description of a patient with Rad50 deficiency characterized by chromosomal instability and defective ATM-dependent signaling. We report here that ATM (defective in ataxia-telangiectasia) phosphorylates Rad50 at a single site (Ser-635) that plays an important adaptor role in signaling for cell cycle control and DNA repair. Although a Rad50 phosphosite-specific mutant (S635G) supported normal activation of ATM in Rad50-deficient cells, it was defective in correcting DNA damage-induced signaling through the ATM-dependent substrate SMC1. This mutant also failed to correct radiosensitivity, DNA double-strand break repair, and an S-phase checkpoint defect in Rad50-deficient cells. This was not due to disruption of the Mre11/Rad50/NBN complex revealing for the first time that phosphorylation of Rad50 plays a key regulatory role as an adaptor for specific ATM-dependent downstream signaling through SMC1 for DNA repair and cell cycle checkpoint control in the maintenance of genome integrity.

Human RAD50 deficiency in a Nijmegen breakage syndrome-like disorder.

The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, 'bird-like' face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell-cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD).

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage.

A defective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.

Metabolic Stress and Mitochondrial Dysfunction in Ataxia-Telangiectasia.

The ataxia-telangiectasia mutated (ATM) protein kinase is, as the name implies, mutated in the human genetic disorder ataxia-telangiectasia (A-T). This protein has its "finger in many pies", being responsible for the phosphorylation of many thousands of proteins in different signaling pathways in its role in protecting the cell against a variety of different forms of stress that threaten to perturb cellular homeostasis. The classical role of ATM is the protection against DNA damage, but it is evident that it also plays a key role in maintaining cell homeostasis in the face of oxidative and other forms of non-DNA damaging stress. The presence of ATM is not only in the nucleus to cope with damage to DNA, but also in association with other organelles in the cytoplasm, which suggests a greater protective role. This review attempts to address this greater role of ATM in protecting the cell against both external and endogenous damage.

Impaired endoplasmic reticulum-mitochondrial signaling in ataxia-telangiectasia.

There is evidence that ATM mutated in ataxia-telangiectasia (A-T) plays a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that ATM-deficient cells are exquisitely sensitive to nutrient deprivation, which can be explained by defective cross talk between the endoplasmic reticulum (ER) and the mitochondrion. Tethering between these two organelles in response to stress was reduced in cells lacking ATM, and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically when compared with control cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells. Our findings reveal that ER-mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion defective in response to nutrient stress, can account for at least part of the mitochondrial dysfunction observed in A-T cells.

Cancer signaling pathways with a therapeutic approach: An overview in epigenetic regulations of cancer stem cells.

One of the most important issues in cancer progression is caner stem cells (CSCs) which have illustrated that the bulk tumors can arise from a special combination of cells. Remarkably, it has been proposed to be a notable and strong factor in carcinogenesis and tumorogenesis and also is a key parameter of therapeutic resistance. In this way, recent findings have shown the key roles of epigenetic regulations in cancer development.Considerably, epigenetic regulations of gene expression is an active and dynamic process including histone modification, DNA methylation and chromatin remodeling with a reversible trait.Meaningly, recent and novel findings have described the significance of epigenetic regulatory proteins from divers features comprising tumorogenesis,stem cell proliferation and carcinogenesis. Evidently, abnormal epigenetic regulations is directly related with many serious disorders particularly different cancers. We here review a discussion of how the deregulation of eclectic pathways containing Sonic Hedgehog (SHH), WNT, Beta catenin and NOTCH can help to carcinogenesis specially focusing to survival and maintenance of CSCs in therapeutic approach.

Rats with a missense mutation in Atm display neuroinflammation and neurodegeneration subsequent to accumulation of cytosolic DNA following unrepaired DNA damage.

Mutations in the ataxia-telangiectasia (A-T)-mutated (ATM) gene give rise to the human genetic disorder A-T, characterized by immunodeficiency, cancer predisposition, and neurodegeneration. Whereas a series of animal models recapitulate much of the A-T phenotype, they fail to present with ataxia or neurodegeneration. We describe here the generation of an Atm missense mutant [amino acid change of leucine (L) to proline (P) at position 2262 (L2262P)] rat by intracytoplasmic injection (ICSI) of mutant sperm into oocytes. Atm-mutant rats (AtmL2262P/L2262P ) expressed low levels of ATM protein, suggesting a destabilizing effect of the mutation, and had a significantly reduced lifespan compared with Atm+/+ Whereas these rats did not show cerebellar atrophy, they succumbed to hind-limb paralysis (45%), and the remainder developed tumors. Closer examination revealed the presence of both dsDNA and ssDNA in the cytoplasm of cells in the hippocampus, cerebellum, and spinal cord of AtmL2262P/L2262P rats. Significantly increased levels of IFN-ß and IL-1ß in all 3 tissues were indicative of DNA damage induction of the type 1 IFN response. This was further supported by NF-κB activation, as evidenced by p65 phosphorylation (P65) and translocation to the nucleus in the spinal cord and parahippocampus. Other evidence of neuroinflammation in the brain and spinal cord was the loss of motor neurons and the presence of increased activation of microglia. These data provide support for a proinflammatory phenotype that is manifested in the Atm mutant rat as hind-limb paralysis. This mutant represents a useful model to investigate the importance of neuroinflammation in A-T.

Phospho-specific antibodies as a tool to study in vivo regulation of BRCA1 after DNA damage.

Phospho-specific antibodies have become very useful reagents for study of signal transduction pathways. This chapter describes the production of phospho-specific antibodies and their use to assess individual phosphorylation events in vivo in cells. The first step involves the synthesis of peptides (12-15 residues), where the phosphorylation site is centrally located, and a cysteine residue is incorporated at either the N- or C-terminus of the peptide to facilitate coupling it to an immunogenic carrier protein. No special immunization protocols are required to generate phospho-specific antibodies. Typically, animals of choice are immunized twice, several weeks apart, and enzyme-linked immunosorbent assay is used to determine the relative titer of sera against phosphorylated and nonphosphorylated peptides. Where the titer against phosphorylated peptides is much greater than nonphosphorylated peptides, the sera can be used at appropriate dilutions without further processing. In case a significant level of antibodies specific to the nonphosphorylated peptide is present in the antisera, an enhancement step is used to obtain a useful phospho-specific antibody. Although these enhanced antisera are suitable for many applications, there may be circumstances where affinity-purified antibodies are required. These antibodies can be used to detect a particular phosphorylation event in vivo using Western blotting, immunoprecipitation, and immunofluorescence.

Dominant negative ATM mutations in breast cancer families.

BACKGROUND: The ATM gene encoding a putative protein kinase is mutated in ataxia-telangiectasia (A-T), an autosomal recessive disorder with a predisposition for cancer. Studies of A-T families suggest that female heterozygotes have an increased risk of breast cancer compared with noncarriers. However, neither linkage analyses nor mutation studies have provided supporting evidence for a role of ATM in breast cancer predisposition. Nevertheless, two recurrent ATM mutations, T7271G and IVS10-6T-->G, reportedly increase the risk of breast cancer. We examined these two ATM mutations in a population-based, case-control series of breast cancer families and multiple-case breast cancer families. METHODS: Five hundred twenty-five or 262 case patients with breast cancer and 381 or 68 control subjects, respectively, were genotyped for the T7271G and IVS10-6T-->G ATM mutations, as were index patients from 76 non-BRCA1/2 multiple-case breast cancer families. Linkage and penetrance were analyzed. ATM protein expression and kinase activity were analyzed in lymphoblastoid cell lines from mutation carriers. All statistical tests were two-sided. RESULTS: In case and control subjects unselected for family history of breast cancer, one case patient had the T7271G mutation, and none had the IVS10-6T-->G mutation. In three multiple-case families, one of these two mutations segregated with breast cancer. The estimated average penetrance of the mutations was 60% (95% confidence interval [CI] = 32% to 90%) to age 70 years, equivalent to a 15.7-fold (95% CI = 6.4-fold to 38.0-fold) increased relative risk compared with that of the general population. Expression and activity analyses of ATM in heterozygous cell lines indicated that both mutations are dominant negative. CONCLUSION: At least two ATM mutations are associated with a sufficiently high risk of breast cancer to be found in multiple-case breast cancer families. Full mutation analysis of the ATM gene in such families could help clarify the role of ATM in breast cancer susceptibility.

Suppression of Tousled-like kinase activity after DNA damage or replication block requires ATM, NBS1 and Chk1.

The human Tousled-like kinases 1 and 2 (TLK) have been shown to be active during S phase of the cell cycle. TLK activity is rapidly suppressed by DNA damage and by inhibitors of replication. Here we report that the signal transduction pathway, which leads to transient suppression of TLK activity after the induction of double-strand breaks (DSBs) in the DNA, is dependent on the presence of a functional ataxia-telangiectasia-mutated kinase (ATM). Interestingly, we have discovered that rapid suppression of TLK activity after low doses of ultraviolet (UV) irradiation or aphidicolin-induced replication block is also ATM-dependent. The nature of the signal that triggers ATM-dependent downregulation of TLK activity after UVC and replication block remains unknown, but it is not due exclusively to DSBs in the DNA. We also demonstrate that TLK suppression is dependent on the presence of a functional Nijmegan Breakage Syndrome protein (NBS1). ATM-dependent phosphorylation of NBS1 is required for the suppression of TLK activity, indicating a role for NBS1 as an adaptor or scaffold in the ATM/TLK pathway. ATM does not phosphorylate TLK directly to regulate its activity, but Chk1 does phosphorylate TLK1 GST-fusion proteins in vitro. Using Chk1 siRNAs, we show that Chk1 is essential for the suppression of TLK activity after replication block, but that ATR, Chk2 and BRCA1 are dispensable for TLK suppression. Overall, we propose that ATM activation is not linked solely to DSBs and that ATM participates in initiating signaling pathways in response to replication block and UV-induced DNA damage.

ATM-Dependent Phosphorylation of All Three Members of the MRN Complex: From Sensor to Adaptor.

The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.

Induced pluripotent stem cells from ataxia-telangiectasia recapitulate the cellular phenotype.

Pluripotent stem cells can differentiate into every cell type of the human body. Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) therefore provides an opportunity to gain insight into the molecular and cellular basis of disease. Because the cellular DNA damage response poses a barrier to reprogramming, generation of iPSCs from patients with chromosomal instability syndromes has thus far proven to be difficult. Here we demonstrate that fibroblasts from patients with ataxia-telangiectasia (A-T), a disorder characterized by chromosomal instability, progressive neurodegeneration, high risk of cancer, and immunodeficiency, can be reprogrammed to bona fide iPSCs, albeit at a reduced efficiency. A-T iPSCs display defective radiation-induced signaling, radiosensitivity, and cell cycle checkpoint defects. Bioinformatic analysis of gene expression in the A-T iPSCs identifies abnormalities in DNA damage signaling pathways, as well as changes in mitochondrial and pentose phosphate pathways. A-T iPSCs can be differentiated into functional neurons and thus represent a suitable model system to investigate A-T-associated neurodegeneration. Collectively, our data show that iPSCs can be generated from a chromosomal instability syndrome and that these cells can be used to discover early developmental consequences of ATM deficiency, such as altered mitochondrial function, that may be relevant to A-T pathogenesis and amenable to therapeutic intervention.

ATM mediated phosphorylation of CHD4 contributes to genome maintenance.

BACKGROUND: In order to maintain cellular viability and genetic integrity cells must respond quickly following the induction of cytotoxic double strand DNA breaks (DSB). This response requires a number of processes including stabilisation of the DSB, signalling of the break and repair. It is becoming increasingly apparent that one key step in this process is chromatin remodelling. RESULTS: Here we describe the chromodomain helicase DNA-binding protein (CHD4) as a target of ATM kinase. We show that ionising radiation (IR)-induced phosphorylation of CHD4 affects its intranuclear organization resulting in increased chromatin binding/retention. We also show assembly of phosphorylated CHD4 foci at sites of DNA damage, which might be required to fulfil its function in the regulation of DNA repair. Consistent with this, cells overexpressing a phospho-mutant version of CHD4 that cannot be phosphorylated by ATM fail to show enhanced chromatin retention after DSBs and display high rates of spontaneous damage. CONCLUSION: These results provide insight into how CHD4 phosphorylation might be required to remodel chromatin around DNA breaks allowing efficient DNA repair to occur.

Evaluation of the role of Finnish ataxia-telangiectasia mutations in hereditary predisposition to breast cancer.

Biallelic mutations in the ataxia-telangiectasia mutated (ATM) gene result in ataxia-telangiectasia (A-T). Studies on A-T families have shown that obligate female carriers have increased risk of developing breast cancer. Here we have evaluated the role of known Finnish ATM germ line mutations as possible breast cancer predisposing alleles outside A-T families by analyzing their prevalence in large cohorts of familial and unselected breast cancer cases. Of seven different alterations, two were observed in the studied breast cancer material. ATM 6903insA (causing protein truncation) was seen in 3/541 familial and 5/1124 unselected cases, but not among healthy population controls (0/1107). 7570G>C (Ala2524Pro) occurred in 1/541 familial and 2/1124 unselected cases compared with 1/1107 in controls. Additionally, 8734A>G (Arg2912Gly) associated previously with breast cancer susceptibility and suggested to be causative also for A-T was detected in 2/541 of familial cases, but not in unselected cases (0/1124) or controls (0/1107). In total, heterozygous ATM mutation carriers were observed in 6/541 familial [P = 0.006, odds ratio (OR) 12.4, 95% confidence interval (CI) 1.5-103.3) and 7/1124 unselected cases (P = 0.07, OR 6.9, 95% CI 0.9-56.4), compared with 1/1107 in controls, suggesting an apparent yet overall limited contribution to predisposition to cancer. The current results also provided evidence for founder effects in the geographical distribution of these mutations. Interestingly, results from functional analysis of the breast cancer-associated ATM mutations indicated that cancer susceptibility is not restricted to mutations with dominant-negative effect on kinase activity, displayed only by 7570G>C, whereas 8734A>G showed only a partial defect in the phosphorylation of ATM substrates, and 6903insA seemed to be a null allele.

Identification of domains of ataxia-telangiectasia mutated required for nuclear localization and chromatin association.

Ataxia-telangiectasia mutated (ATM) is essential for rapid induction of cellular responses to DNA double strand breaks (DSBs). In this study, we mapped a nuclear localization signal (NLS), 385KRKK388, within the amino terminus of ATM and demonstrate its recognition by the conventional nuclear import receptor, the importin alpha1/beta1 heterodimer. Although mutation of this NLS resulted in green fluorescent protein (GFP) x ATM(NLSm) localizing predominantly within the cytoplasm, small amounts of nuclear GFP x ATM(NLSm) were still sufficient to elicit a DNA damage response. Insertion of an heterologous nuclear export signal between GFP and ATM(NLSm) resulted in complete cytoplasmic localization of ATM, concomitantly reducing the level of substrate phosphorylation and increasing radiosensitivity, which indicates a functional requirement for ATM nuclear localization. Interestingly, the carboxyl-terminal half of ATM, containing the kinase domain, which localizes to the cytoplasm, could not autophosphorylate itself or phosphorylate substrates, nor could it correct radiosensitivity in response to DSBs even when targeted to the nucleus by insertion of an exogenous NLS, demonstrating that the ATM amino terminus is required for optimal ATM function. Moreover, we have shown that the recruitment/retention of ATM at DSBs requires its kinase activity because a kinase-dead mutant of GFP x ATM failed to form damage-induced foci. Using deletion mutation analysis we mapped a domain in ATM (amino acids 5-224) required for its association with chromatin, which may target ATM to sites of DNA damage. Combined, these data indicate that the amino terminus of ATM is crucial not only for nuclear localization but also for chromatin association, thereby facilitating the kinase activity of ATM in vivo.