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. 2003 Oct 15;22(20):5612-21.
doi: 10.1093/emboj/cdg541.

Requirement of the MRN complex for ATM activation by DNA damage

Tamar Uziel  1 , Yaniv Lerenthal, Lilach Moyal, Yair Andegeko, Leonid Mittelman, Yosef Shiloh
Affiliations

Requirement of the MRN complex for ATM activation by DNA damage

Tamar Uziel et al. EMBO J. .

Abstract

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.

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Figures

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Fig. 1. Immunoblotting analysis of lymphoblastoid cell lines from patients with various genome instability syndromes. A-TLD is represented by patients with the two variants of this disorder, A-TLD(M) and A-TLD(S) (see text). Note the reduced levels of the Nbs1 and Rad50 in A-TLD patients (Stewart et al., 1999).
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Fig. 2. ATM activation, reflected by its autophosphorylation, in lymphoblastoid lines from genomic instability syndromes. Following NCS treatments, cellular extracts were subjected to immunoblotting analysis using an antibody directed against phosphorylated Ser1981 of ATM. An anti-ATM antibody was used to control for ATM amounts. (A) ATM activation in response to increasing NCS doses (treatment time 15 min). (B) Time course of ATM autophosphorylation following treatment with 5 ng/ml of NCS.
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Fig. 3. Nuclear retention of ATM following radiomimetic treatment in cell lines with different genotypes. (A) Biochemical demonstration of damage-induced nuclear retention of ATM, reflected as increased resistance of this protein to detergent extraction. Lymphoblastoid cells were treated with the indicated doses of NCS, and protein extracts underwent successive fractionations with detergents. ATM was visualized in the resultant fractions using immunoblotting analysis. The extraction-resistant fraction of ATM (‘retained ATM’) represents a portion of ATM that adheres to DSB sites in a dose-dependent manner (see top panel representing a normal control). ‘Non-retained ATM’ represents a clarified cell extract from the first fractionation that contains unbound ATM in each sample. Dose-dependent nuclear retention of ATM is abrogated in A-TLD cells in correlation with the extent of Mre11 deficiency. Note intermediate impairment of ATM retention in the NBS cell line. (B) Demonstration of nuclear retention of ATM in lymphoblast cells using in situ detergent extraction followed by immunostaining (Andegeko et al., 2001). In untreated cells most of the nuclear content of ATM is extracted and washed out, while prior NCS treatment (50 ng/ml) renders a significant portion of nuclear ATM extraction-resistant. This process is impaired to different extents in A-TLD and NBS cells, most seriously in the severe A-TLD variant. (C) Quantitation of the amount of ATM fluorescence in lymphoblast cells after treatment with 50 ng/ml of NCS and subsequent detergent extraction in situ. The fluorescent signal was quantitated in at least 16 cells for each point.
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Fig. 4. Phosphorylation of ATM effectors in lymphoblastoid cells following treatment with 10 ng/ml of NCS. Top panel: phosphorylation of the Chk2 kinase reflected as retarded electrophorectic migration of the phosphorylated protein. Note Nbs1 dependence of this phosphorylation at the treatment dose used in this experiment, the delayed and reduced phosphorylation of Chk2 in A-TLD(M) cells, and the lack of phosphorylation in A-TLD(S) cells. Middle panel: phosphorylation of p53 on Ser15 detected by a phospho-specific antibody. A moderate retardation of this phosphorylation is noticed in NBS cells, and the response is attenuated in the A-TLD variants. Bottom panel: phosphorylation of Hdm2 on Ser395. This phosphorylation abolishes the immunoreactivity of Hdm2 with the monoclonal antibody 2A10 and is therefore expressed as loss of the Hdm2 band on immunoblots (Khosravi et al., 1999). The band below the Hdm2 band represents a cross-reacting protein and conveniently serves as a loading control. Hdm2 phosphorylation occurs at a normal rate in NBS cells, is moderately impaired in A-TLD(M) cells, and is abolished in A-TLD(S) cells.
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Fig. 5. Integrative visualization of phosphorylation of ATM substrates in human fibroblast lines in response to DSBs, visualized by immunostaining with an antibody raised against phosphorylated ATM target sequence (‘anti-phospho-[SQ/TQ]’). (A) Thirty minutes following NCS treatment, a vigorous response is observed in wild-type cells while the diffuse background staining in A-T cells is barely changed. Phase contrast images of the cells indicate that this response is confined to the nucleus. (B) Phosphorylation of ATM substrates in fibroblast cell lines with various genotypes following treatment with 50 ng/ml of NCS. Fluorescence intensity after staining with the anti-phospho-(SQ/TQ) antibody was averaged over ∼20 cells for each genotype and treatment. The numbers below the line represent the ratios between fluorescence values after NCS treatment and without treatment. Note the similar lack of response in A-T and A-TLD(S) cells and the variably low responses of all other genotypes. In some cell lines initial high level of phosphorylation is noticed. This constitutive damage response is occasionally observed in cells with defective damage response and represents elevated basal levels of DNA damage (Gatei et al., 2001; Kamsler et al., 2001).
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Fig. 6. Reconstitution of the MRN complex and ATM activation in A-TLD(S) and NBS cells by ectopic expression of Mre11 and Nbs1, respectively. (A) Immunofluorescence images of the three components of the MRN complex in hTERT-immortalized A-TLD(S) fibroblasts following transduction with a control vector expressing GFP and a vector expressing wild-type Mre11 protein. In each panel the same field is shown in all photographs. (B) Immunoblotting analysis showing ATM activation in A-TLD(S) cells transduced with the control and Mre11 vectors. Note reconstitution of endogenous Rad50 and Nbs1 levels following ectopic expression of Mre11 and reconstitution of damage-induced ATM activation. (C) ATM activation in NBS cells reconstituted by ectopic expression of Nbs1 (Tauchi et al., 2001).
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Fig. 7. Inability of nuclease-defective Mre11 to fully reconstitute ATM activation in A-TLD(S) cells. (A) Expression of recombinant, mutant Mre11 and endogenous Rad50 and Nbs1 in A-TLD(S) fibroblasts transduced with GFP and Mre11-3 vectors. Note reconstitution of endogenous Rad50 and Nbs1 levels and nuclear localization. (B) Visualization of ATM activation in the same cells by immunostaining with the antibody against phosphorylated Ser1981. Note the defective ATM activation in cells transduced with the Mre11-3 mutant.
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Fig. 8. A model depicting early events in the cellular response to moderate DSB levels. The MRN complex is essential for the initial damage processing. Processed DNA lesions lead to the recruitment and activation of ATM, which in turn phosphorylates its substrates, among them Nbs1 and Mre11. Phosphorylated Nbs1 facilitates the phosphorylation of certain ATM substrates and plays a role in the activation of cell cycle checkpoint. It is not clear whether Nbs1 acts alone in this capacity or in the context of the MRN complex.
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