Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox-active cofactor in many biological processes, including DNA replication and repair. Eukaryotic ribonucleotide reductases (RNRs) are Fe-dependent enzymes that catalyze deoxyribonucleoside diphosphate (dNDP) synthesis. We show here that the levels of the Sml1 protein, a yeast RNR large-subunit inhibitor, specifically decrease in response to both nutritional and genetic Fe deficiencies in a Dun1-dependent but Mec1/Rad53-and Aft1-independent manner. The decline of Sml1 protein levels upon Fe starvation depends on Dun1 forkheadassociated and kinase domains, the 26S proteasome, and the vacuolar proteolytic pathway. Depletion of core components of the mitochondrial iron-sulfur cluster assembly leads to a Dun1-dependent diminution of Sml1 protein levels. The physiological relevance of Sml1 downregulation by Dun1 under low-Fe conditions is highlighted by the synthetic growth defect observed between dun1⌬ and fet3⌬ fet4⌬ mutants, which is rescued by SML1 deletion. Consistent with an increase in RNR function, Rnr1 protein levels are upregulated upon Fe deficiency. Finally, dun1⌬ mutants display defects in deoxyribonucleoside triphosphate (dNTP) biosynthesis under low-Fe conditions. Taken together, these results reveal that the Dun1 checkpoint kinase promotes RNR function in response to Fe starvation by stimulating Sml1 protein degradation.
Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the de novo synthesis of deoxyribonucleoside diphosphates (dNDPs), which are the precursors for DNA replication and repair. Eukaryotic RNRs are comprised of ␣ and  subunits that form an active quaternary structure, (␣ 2 ) 3 ( 2 ) m , where m is 1 or 3. ␣ 2 , referred to as the large or R1 subunit, contains the catalytic and allosteric sites, and  2 , known as the small or R2 subunit, harbors a diferric center that is responsible for generating and keeping a tyrosyl radical required for catalysis (reviewed in references 1 to 3). In the budding yeast Saccharomyces cerevisiae, the large R1 subunit is formed by an Rnr1 homodimer and the small R2 subunit is composed of an Rnr2-Rnr4 heterodimer (reviewed in reference 4). Eukaryotic cells tightly control RNR activity to achieve adequate and balanced deoxyribonucleoside triphosphate (dNTP) pools that ensure accurate DNA synthesis and genomic integrity. In response to DNA damage or DNA replication stress or when cells enter S phase of the cell cycle, the yeast Mec1/Rad53/Dun1 checkpoint kinase cascade activates RNR function (reviewed in reference 4). Briefly, genotoxic stress activates Mec1, which phosphorylates and enhances Rad53 kinase activity (5, 6). A diphosphothreonine motif in hyperphosphorylated Rad53 protein is subsequently recognized by Dun1's forkhead-associated (FHA) domain, leading to Rad53-mediated phosphorylation and activation of Dun1 kinase (7-11), which promotes RNR function through multiple mechanisms. One mechanism involves the transcriptional repressor Crt1,...