The Conserved Mec1/Rad53 Nuclear Checkpoint Pathway Regulates Mitochondrial DNA Copy Number inSaccharomyces cerevisiae

Abstract

How mitochondrial DNA (mtDNA) copy number is determined and modulated according to cellular demands is largely unknown. Our previous investigations of the related DNA helicases Pif1p and Rrm3p uncovered a role for these factors and the conserved Mec1/Rad53 nuclear checkpoint pathway in mtDNA mutagenesis and stability in Saccharomyces cerevisiae. Here, we demonstrate another novel function of this pathway in the regulation of mtDNA copy number. Deletion of RRM3 or SML1, or overexpression of RNR1, which recapitulates Mec1/Rad53 pathway activation, resulted in an approximately twofold increase in mtDNA content relative to the corresponding wild-type yeast strains. In addition, deletion of RRM3 or SML1 fully rescued the ϳ50% depletion of mtDNA observed in a pif1 null strain. Furthermore, deletion of SML1 was shown to be epistatic to both a rad53 and an rrm3 null mutation, placing these three genes in the same genetic pathway of mtDNA copy number regulation. Finally, increased mtDNA copy number via the Mec1/Rad53 pathway could occur independently of Abf2p, an mtDNA-binding protein that, like its metazoan homologues, is implicated in mtDNA copy number control. Together, these results indicate that signaling through the Mec1/Rad53 pathway increases mtDNA copy number by altering deoxyribonucleoside triphosphate pools through the activity of ribonucleotide reductase. This comprises the first linkage of a conserved signaling pathway to the regulation of mitochondrial genome copy number and suggests that homologous pathways in humans may likewise regulate mtDNA content under physiological conditions. INTRODUCTIONSince the discovery that the mitochondrial genome is present at multiple copies per cell (100 -10,000 in humans) and is subject to dynamic regulation with regard to tissue type, metabolic signals, and environmental stimuli, an understanding of the pathways and mechanisms that regulate cellular mitochondrial DNA (mtDNA) copy number has been sought (Moraes, 2001). Although nuclear gene products that have direct roles in mtDNA replication or stability have been implicated in copy number regulation (Schultz et al., 1998;Zelenaya-Troitskaya et al., 1998;Ekstrand et al., 2004;Matsushima et al., 2004;Tyynismaa et al., 2004), signaling pathways involved in modulating cellular mtDNA content have not been fully elucidated. However, important insight into mtDNA copy number regulation and its clinical significance has been gleaned from the study of human mitochondrial disease patients. For example, several mtDNA-depletion syndromes have been characterized, the hallmark of which is decreased mtDNA copy number and/or integrity in certain tissues (Moraes et al., 1991;Elpeleg et al., 2002). Identification of the nuclear genetic defects underlying several of these diseases has underscored a critical role for cellular deoxyribonucleoside triphosphate (dNTP) pools in mtDNA copy number and stability. For example, mutations in the mitochondrial thymidine kinase gene cause mitochondrial depletion myopathy (Saada et al., 2001), mutations...

Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

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Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

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Mitochondrial fission proteins Fis1 and Mdv1, but not Dnm1, play a role in maintenance of heteroplasmy in budding yeast

“…The a-WT and a-WT strains were cultivated in YPD medium only when the effect of glucose repression was tested. Total cellular DNA was extracted and used as the template for quantitative real-time PCR in a LightCycler Ò 480 (Roche) as described [23].…”

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