The fidelity of DNA replication and repair processes is critical for maintenance of genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production and thus plays an essential role in DNA synthesis. The level and activity of RNR are highly regulated by the cell cycle and DNA damage checkpoints, which maintain optimal dNTP pools required for genetic fidelity. RNRs are composed of a large subunit that binds the nucleoside diphosphate substrates and allosteric effectors and a small subunit that houses the di-iron tyrosyl radical cofactor essential for the reduction process. In Saccharomyces cerevisiae, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). Here we report the subcellular localization of Rnr1-4 during normal cell growth and the redistribution of Rnr2 and Rnr4 in response to DNA damage and replicational stress. During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm and Rnr2 and Rnr4 are predominantly present in the nucleus. Under genotoxic stress, Rnr2 and Rnr4 become redistributed to the cytoplasm in a checkpoint-dependent manner. Subcellular redistribution of Rnr2 and Rnr4 can occur in the absence of the transcriptional induction of the RNR genes after DNA damage and likely represents a posttranslational event. These results suggest a mechanism by which DNA damage checkpoint modulates RNR activity through the temporal and spatial regulation of its subunits. E ukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to respond to genotoxic stress by arresting the cell cycle and inducing the transcription of genes that facilitate repair (1, 2). Failure of DNA damage response can result in genomic instability and cancer predisposition (3, 4). In mammalian cells the protein kinases ATM, ATR, and CHK2 are crucial for activating signaling pathways for cell survival after DNA damage (5-7). In the yeast Saccharomyces cerevisiae, the ATR homologue Mec1 and CHK2 homologue Rad53 are key regulators of cellular response to DNA damage, controlling the G 1 , S, and G 2 cell cycle checkpoints as well as transcriptional induction (8). Dun1, a protein kinase similar to Rad53, is also involved in these processes (9, 10). Among the best-studied transcriptional targets of the Mec1͞ Rad53͞Dun1 checkpoint pathway are the genes encoding ribonucleotide reductase (RNR; refs. 9 and 11-13).The enzymatic activity of RNR depends on the formation of a complex between two different subunits, R1 and R2. The large subunit R1 is a dimer and contains the active site for reduction of nucleoside diphosphate (NDP) substrates and the effector sites that control substrate specificity and enzymatic activity. The small subunit R2 is also a dimer that houses the di-iron tyrosyl radical (Y⅐) cofactor essential for NDP reduction. The active form of RNR is proposed to be a 1:1 complex of R1 and R2 (14-16).In budding yeast there are four RNR genes, two that code for a large subunit (RNR1 and RNR3) and two that code for a s...

Background: Yeast RNR small subunit is an Rnr2-Rnr4 heterodimer; only Rnr2 contains a cluster. Results: rnr4 and dre2 mutants are defective in Rnr2 cluster formation and display synthetic growth defects with grx3/4. Conclusion: Rnr4 stabilizes Rnr2 for cluster assembly via a pathway dependent on monothiol glutaredoxins Grx3/Grx4 and Fe-S cluster protein Dre2. Significance: Understanding RNR cluster assembly may provide new cancer therapeutic strategy.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (␣, oligomeric state unknown) and a small subunit R2 (␤ 2). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe) 2-Y⅐] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (␤␤ ), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, ␤␤ is redistributed from the nucleus to the cytoplasm in a checkpointdependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in ␤␤ nuclear localization: the importin ␤ homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of ␤␤ nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with ␤␤ . Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of ␤␤ by Kap122 or as an anchor to retain ␤␤ in the nucleus. DNA-damage checkpoint ͉ subcellular redistributionT he levels and relative ratios of dNTP pools are important for high-fidelity DNA replication and repair (1). Failure to increase dNTP levels at the G 1 -to-S transition of the cell cycle is a lethal event at cellular level (2, 3). Conversely, elevated dNTP pools throughout the cell cycle lead to increased mutation rates (4-6). Imbalance in dNTP pools also contributes to mutagenesis by reducing the fidelity of DNA polymerases (7-9). Eukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to ensure proper dNTP pool sizes during the normal cell-cycle progression and in response to genotoxic stress (3, 10-14). A major target of such checkpoint regulation is ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate, an essential step in de novo biosynthesis of dNTPs (15).Class I RNRs were identified originally in Escherichia coli and are conserved from yeast to mammal (16). The mechanisms of enzymatic catalysis (17) and allosteric regulation (18, 19) have been studied extensively in E. coli and, more recently, in mice (20, 21). The archetype RNR holoenzyme consists of a large subunit R1 (␣, whose oligomeric state in eukaryotes is not completely understood) (22) and a homodimeric small subunit (␤ 2 ) (20). The eukaryotic R1 contains the catalytic site, an effector site that controls substrate specificity, an activity site that controls turnover, and a weak ATP-binding site that cont...

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,...

Eukaryotic ribonucleotide reductases (RNRs) require a diferrictyrosyl radical (Fe III 2 -Y•) cofactor to produce deoxynucleotides essential for DNA replication and repair. This metallocofactor is an important target of RNR-based therapeutics, although mechanisms of in vivo cofactor assembly, inactivation, and reactivation are poorly understood. Here, we demonstrate that the conserved Fe-S protein-diflavin reductase complex, Dre2-Tah18, plays a critical role in RNR cofactor biosynthesis. Depletion of Dre2 affects both RNR gene transcription and mRNA turnover through the activation of the DNA-damage checkpoint and the Aft1/Aft2-controlled iron regulon. Under conditions of comparable RNR protein levels, cells with diminishing Dre2 have significantly reduced ability to make deoxynucleotides. Furthermore, the kinetics and levels of in vivo reconstitution of the RNR cofactor are severely impaired in two conditional tah18 mutants. Together, these findings provide insight into RNR cofactor formation and reveal a shared mechanism underlying assembly of the Fe

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (α) that contains the catalytic site and RR2 (β) that houses a diferrictyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP–CDP [AMPPNP, adenosine 5-(β,γ-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.

Ribonucleotide reductase (RNR) catalyzes the rate-liming step in de novo deoxyribonucleotide biosynthesis and is essential in DNA replication and repair. Cells have evolved complex mechanisms to modulate RNR activity during normal cell cycle progression and in response to genotoxic stress. A recently characterized mode of RNR regulation is DNA damage-induced RNR subunit redistribution. The RNR holoenzyme consists of a large subunit, R1, and a small subunit, R2. The Saccharomyces cerevisiae R2 is an Rnr2:Rnr4 heterodimer. Rnr2 generates a diferric-tyrosyl radical cofactor required for catalysis; Rnr4 facilitates cofactor assembly and stabilizes the resulting holo-heterodimer. Upon DNA damage, Rnr2 and Rnr4 undergo checkpoint-dependent, nucleus-to-cytoplasm redistribution, resulting in colocalization of R1 and R2. Here we present evidence that Rnr2 and Rnr4 are transported between the nucleus and the cytoplasm as one protein complex. Tagging either Rnr2 or Rnr4 with a nuclear export sequence causes cytoplasmic localization of both proteins. Moreover, mutations at the Rnr2:Rnr4 heterodimer interface can affect the localization of both proteins without disrupting the heterodimeric complex. Finally, the relocalization of Rnr4 appears to involve both active export and blockage of nuclear import. Our findings provide new insights into the mechanism of DNA damage-induced RNR subunit redistribution.

Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function. Ribonucleotide reductase (RNR)5 catalyzes the rate-limiting step in the de novo deoxyribonucleotide (dNTP) synthesis by converting ribonucleoside diphosphates to the corresponding deoxy forms. In eukaryotes, the RNR holoenzyme is composed of a large or R1 subunit that contains the catalytic and allosteric sites, and a small or R2 subunit that harbors a di-iron center, which is responsible for generating and keeping a tyrosyl radical required for catalysis (reviewed in Refs.