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Vol. 16, Issue 6, 3010-3018, June 2005

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

Sean D. Taylor ^* , Hong Zhang  , Jana S. Eaton ^*  , Matthew S. Rodeheffer  ^|| ¶, Maria A. Lebedeva ^*, Thomas W. O'Rourke , Wolfram Siede ^#, and Gerald S. Shadel ^*

^* Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023; Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322; ^# Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107; ^|| Graduate Program in Biochemistry and Cell and Developmental Biology, Emory University, Atlanta, GA 30322; and Graduate Program in Genetics and Molecular Biology, Emory University, Atlanta, GA 30322

Submitted January 19, 2005; Revised March 9, 2005; Accepted March 31, 2005
Monitoring Editor: Thomas Fox

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 50% 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.

Abbreviations used: dNTP, deoxyribonucleoside triphosphate; mtDNA, mitochondrial DNA; RNR, ribonucleotide reductase.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

These authors contributed equally to this work.

^¶ Present address: Laboratory of Molecular Genetics, The Rockefeller University, 1230 York Ave., Box 305, New York, NY 10021.

Address correspondence to: Gerald S. Shadel (gerald.shadel{at}yale.edu).

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