Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis
Jana S. Eaton, Z. Ping Lin, Alan C. Sartorelli, Nicholas D. Bonawitz, Gerald S. Shadel
Jana S. Eaton, Z. Ping Lin, Alan C. Sartorelli, Nicholas D. Bonawitz, Gerald S. Shadel
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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 2

RR and ATM are required for increased mtDNA copy number and biogenesis in response to IR and proper regulation of RR subunit expression.

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RR and ATM are required for increased mtDNA copy number and biogenesis i...
(A) Relative mtDNA levels (plotted as in Figure 1) in primary wild-type fibroblasts 24 and 48 hours after exposure to 17.5 Gy of IR with all values compared with nonirradiated, untreated wild-type cells at 24 hours. Cells were exposed to 1 μM Triapine for 24 hours either immediately after IR (24 hours) or after a 24-hour recovery (48 hours) as indicated. The mean ± SEM is plotted. A 1-way ANOVA was used to determine statistical significance as indicated. (B) Relative mtDNA copy number of wild-type and A-T primary fibroblasts 24 and 48 hours after exposure (+) to 17.5 Gy of IR is plotted as in Figure 1. (C) Mitochondrial mass of wild-type and A-T patient fibroblasts 48 hours after IR. Median MitoTracker Green FM fluorescence intensity (mean ± SEM) is plotted. FACS histograms from 1 representative replicate are shown in the right panels. The percentage of cells collected (% max; y axis) with the indicated amount of fluorescence on the x axis (log scale) is shown. (D) Western blot analysis of R1, R2, and p53R2 (p2) from nonirradiated A-T cells (C) or WT and A-T cells 48 hours after 17.5 Gy (IR) from a representative experiment. Actin was probed as a control (C) for protein loading (the relative amount of actin in each lane is shown below the actin panel; wild-type nonirradiated was set to 100%). Actin-normalized signals from 3 independent experiments are depicted graphically on the right of the panels with the wild-type nonirradiated control protein levels set to 1 as indicated by the dotted line.