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...