Abstract
The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. Previously, we found that decreased histone expression induces mitochondrial respiration, raising the question whether the DDR also stimulates respiration. Here, using oxygen consumption and ATP assays, RT-qPCR and ChIP-qPCR methods, and dNTP analyses, we show that DDR activation in the budding yeast Saccharomyces cerevisiae, either by genetic manipulation or by growth in the presence of genotoxic chemicals, induces respiration. We observed that this induction is conferred by reduced transcription of histone genes and globally decreased DNA nucleosome occupancy. This globally altered chromatin structure increased the expression of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain, oxidative phosphorylation, elevated oxygen consumption, and ATP synthesis. The elevated ATP levels resulting from DDR-stimulated respiration drove enlargement of dNTP pools; cells with a defect in respiration failed to increase dNTP synthesis and exhibited reduced fitness in the presence of DNA damage. Together, our results reveal an unexpected connection between respiration and the DDR and indicate that the benefit of increased dNTP synthesis in the face of DNA damage outweighs possible cellular damage due to increased oxygen metabolism.
Highlights
The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival
Phosphorylation of Dif1p by Dun1p releases Rnr2p and Rnr4p chromatin immunoprecipitation coupled with quantitative PCR; ETC, electron transport chain; 4-NQO, 4-nitroquinoline 1-oxide; OXPHOS, oxidative phosphorylation; RNR, ribonucleotide reductase; ROS, reactive oxygen species; RT-qPCR, real-time reverse transcription quantitative PCR; YPD, yeast extract/peptone/dextrose; TCA, tricarboxylic acid; MMS, methylmethane sulfonate; pol, polymerase
Our data show that DDR and growth in the presence of sublethal concentrations of genotoxic chemicals activate respiration to increase ATP production and to elevate deoxyribonucleoside triphosphate (dNTP) levels, which are required for efficient DNA repair and cell survival upon DNA damage
Summary
The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. Using oxygen consumption and ATP assays, RT-qPCR and ChIP-qPCR methods, and dNTP analyses, we show that DDR activation in the budding yeast Saccharomyces cerevisiae, either by genetic manipulation or by growth in the presence of genotoxic chemicals, induces respiration. We observed that this induction is conferred by reduced transcription of histone genes and globally decreased DNA nucleosome occupancy. This globally altered chromatin structure increased the expression of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain, oxidative phosphorylation, elevated oxygen consumption, and ATP synthesis. During S phase or after DNA damage, Dun1p phosphorylates and induces degradation of Sml1p, a protein that binds and inhibits the Rnr1p subunit (Fig. 1) [31,32,33,34]
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