Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis
Jana S. Eaton, … , Nicholas D. Bonawitz, Gerald S. Shadel
Jana S. Eaton, … , Nicholas D. Bonawitz, Gerald S. Shadel
Published September 4, 2007
Citation Information: J Clin Invest. 2007;117(9):2723-2734. https://doi.org/10.1172/JCI31604.
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Research Article Genetics

Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis

Abstract

Ataxia-telangiectasia mutated (ATM) kinase orchestrates nuclear DNA damage responses but is proposed to be involved in other important and clinically relevant functions. Here, we provide evidence for what we believe are 2 novel and intertwined roles for ATM: the regulation of ribonucleotide reductase (RR), the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates, and control of mitochondrial homeostasis. Ataxia-telangiectasia (A-T) patient fibroblasts, wild-type fibroblasts treated with the ATM inhibitor KU-55933, and cells in which RR is inhibited pharmacologically or by RNA interference (RNAi) each lead to mitochondrial DNA (mtDNA) depletion under normal growth conditions. Disruption of ATM signaling in primary A-T fibroblasts also leads to global dysregulation of the R1, R2, and p53R2 subunits of RR, abrogation of RR-dependent upregulation of mtDNA in response to ionizing radiation, high mitochondrial transcription factor A (mtTFA)/mtDNA ratios, and increased resistance to inhibitors of mitochondrial respiration and translation. Finally, there are reduced expression of the R1 subunit of RR and tissue-specific alterations of mtDNA copy number in ATM null mouse tissues, the latter being recapitulated in tissues from human A-T patients. Based on these results, we propose that disruption of RR and mitochondrial homeostasis contributes to the complex pathology of A-T and that RR genes are candidate disease loci in mtDNA-depletion syndromes.

Authors

Jana S. Eaton, Z. Ping Lin, Alan C. Sartorelli, Nicholas D. Bonawitz, Gerald S. Shadel

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Figure 3

ATM is required for mtDNA maintenance and normal expression of RR subunits in the absence of DNA damage.

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ATM is required for mtDNA maintenance and normal expression of RR subuni...
(A) Relative mtDNA copy number (plotted as in Figure 1) of wild-type and A-T primary fibroblasts at 24 or 48 hours. The mean ± SEM is plotted with significant statistical differences via Student’s t test indicated. (B) Relative mtDNA copy number of wild-type primary fibroblasts untreated (C) or treated for 24 hours with indicated concentration of ATM inhibitor KU-55933, after a 48-hour preincubation. The mean ± SEM is plotted with significant statistical differences via Student’s t test indicated. (C) Western blot analysis of R1, R2, and p53R2 (p2) of wild-type and A-T primary fibroblasts at 24 and 48 hours from a representative blot corresponding to conditions in A as depicted in Figure 2D. (D) Western blot analysis of R1, R2, and p53R2 of wild-type primary fibroblasts treated with 10 μM ATM inhibitor KU-55933 (ATMi) as described in B and depicted as in Figure 2D. (E) Relative levels of BACE2-normalized R1 and p53R2 mRNA transcript levels in A-T patient–derived fibroblasts. The mean ± SEM from 3 independent experiments is plotted. (F) Relative fluorescence intensity of dihydroethidium, a dye that detects cellular ROS, in wild-type and A-T primary fibroblasts at 24 hours (proliferating) or 6 days (confluent) with wild-type fluorescence arbitrarily set to 1.0 at each time point. The mean ± SEM of 1 representative experiment is plotted with significant statistical differences via Student’s t test indicated.