Dif1 controls subcellular localization of ribonucleotide reductase by mediating nuclear import of the R2 subunit.

Maintenance of genomic stability depends on faithful replication of DNA and repair of lesions after damage. Fidelity of both DNA replication and repair is influenced by perturbation in the sizes and relative ratios of cellular deoxynucleotide triphosphate (dNTP) pools. Ribonucleotide reductase (RNR) catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms and is largely responsible for maintaining cellular dNTP pools (34). As maximal DNA synthesis requires high concentrations of dNTPs, the RNR activity plays an important role in cell proliferation (31). On the other hand, increased RNR activity has been associated with malignant transformation and resistance to chemotherapy (12, 14, 25, 56). The class I RNR holoenzymes are commonly found in eukaryotes and eubacteria and comprise two subunits, R1 and R2 (34). The active site and multiple binding sites for allosteric effectors reside in R1 (20), which can exist as a dimer, tetramer, and hexamer depending on the nucleotides present and their concentrations (21, 37, 47). R2 is a homodimer or heterodimer that houses a diferric-tyrosyl radical cofactor [(Fe)2-Y] essential for nucleotide reduction (36, 40, 42). Mammalian genomes contain a single R1 gene and two R2 genes; the cell cycle-regulated RRM2 is responsible for providing dNTPs in actively dividing cells, and the DNA damage-inducible p53R2 is required for replenishing dNTP pools in cells under genotoxic stress (9, 22, 44). Loss of p53R2 causes mitochondrial DNA depletion and increased apoptosis (22). The budding yeast Saccharomyces cerevisiae has two R1 genes, RNR1 and RNR3. RNR1 is essential for mitotic growth, while RNR3 is highly inducible after DNA damage but dispensable for cell viability. The yeast R2 is a heterodimer of Rnr2 and Rnr4 (17, 36, 41). Only Rnr2 is capable of forming the (Fe)2-Y cofactor (36). Rnr4 is incapable of forming any radical, as it contains substitutions in three of the six conserved residues required for iron binding (17, 35, 48). Nevertheless, Rnr4 is required to facilitate the generation of radicals in Rnr2 and stabilizes the resulting heterodimer both in vitro and in vivo (7, 36, 46). Because of its central role in generation and maintenance of dNTP pools, the RNR enzyme is subjected to complex regulation both in cells going through normal cell cycle progression and in cells (resting or proliferating) encountering genotoxic stress. RNR concentrations and activity can be modulated at the level of transcription, protein inhibitor interaction, and protein degradation, as well as subcellular localization, all of which are under the control of the DNA damage and replication checkpoint kinases ATR/Mec1 and CHK2/Rad53. Transcription of the mammalian p53R2 gene is induced by UV irradiation in a p53-dependent manner (32, 44). In S. cerevisiae, DNA damage and replication blockage induce transcription of three of the four RNR genes (RNR2 to RNR4) through checkpoint kinase-mediated phosphorylation and removal of the transcriptional repressor Crt1 from its target promoters (18). The S. cerevisiae genome encodes the 104-residue protein Sml1 that binds and inhibits the R1 subunit (8, 54). Sml1 is an unstable protein and undergoes checkpoint-dependent phosphorylation and degradation during S phase of the cell cycle and in cells experiencing genotoxic stress (52, 55). Although no apparent sequence homolog of Sml1 has been identified in multicellular organisms, Sml1 can bind the mammalian R1 and inhibits its activity (8, 53), suggesting a conserved mechanism of Sml1-R1 interaction and inhibition. Dynamic change in subcellular localization patterns of the R2 subunit offers another major mode of RNR regulation. In both S. cerevisiae and Schizosaccharomyces pombe, R1 is constitutively localized in the cytoplasm, whereas R2 is predominantly localized in the nucleus except for S phase of the cell cycle, when R2 becomes colocalized with R1 in the cytoplasm (29, 50). Upon DNA damage or replication blockage, R2 is redistributed from the nucleus to the cytoplasm in a checkpoint-dependent manner, leading to colocalization of R1 and R2 (29, 50). A similar dynamic change in RNR subunit subcellular localization has also been reported in plant cells and mammalian cells (28, 30), although it is unclear whether the mechanistic details of the localization changes are conserved throughout evolution. In S. cerevisiae, the heterodimeric R2 subunit Rnr2-Rnr4 is cotransported between the nucleus and the cytoplasm (2). Proper nuclear localization of R2 requires the WD40 protein Wtm1, which acts as a nuclear anchor of R2, and the karyopherin Kap122, which is involved in importing Wtm1 into the nucleus (26, 51). Subcellular localization of S. pombe R2 is controlled by the 127-residue protein Spd1 (29). Spd1 was originally identified in a screen for S-phase inhibitors because its overexpression causes G1 arrest (49). Spd1 is subjected to checkpoint-dependent phosphorylation and proteolysis in response to DNA damage (4, 29). Its destruction has been found to correlate with redistribution of R2 from the nucleus to the cytoplasm (29, 43). Overexpression of Spd1 retains R2 in the nucleus even in the presence of DNA damage. Thus, Spd1 likely acts as a nuclear anchor of R2 (29). The S. pombe Spd1 and S. cerevisiae Sml1 proteins share no sequence homology despite their similarities in protein sizes, functional roles in RNR inhibition, and regulation by the checkpoint kinases. Sml1 inhibits RNR through binding of the R1 subunit, whereas Spd1 inhibits RNR through sequestration of the R2 subunit in the nucleus away from R1. Interestingly, purified recombinant Spd1 protein has been shown to bind the S. pombe R1 subunit and inhibits its activity in vitro, while Spd1-R2 interaction cannot be detected under the same conditions (16). However, the measured specific activity of purified S. pombe R1 in the report (∼10 nmol dCDP/mg/min [16]) is extremely low relative to that of the S. cerevisiae R1 (250 nmol dCDP/mg/min [16]; ∼800 nmol dCDP/mg/min [35]) and the mouse R1 (130 nmol dCDP/mg/min [16]), suggesting low levels of active S. pombe R1 proteins in the preparation. Hence, the mechanistic basis for Spd1-mediated RNR inhibition remains to be elucidated. In this study, we identified an S. cerevisiae gene encoding a protein with sequence homology to both Sml1 and Spd1. Since the submission of the manuscript, the same gene was named as DIF1 (damage-regulated import factor) by an independent study because of its role in nuclear import of R2 and its regulation by the DNA damage checkpoint (27). DIF1 orthologs are found in multiple fungal genomes. We demonstrate that Dif1 is required for proper nuclear localization of the R2 subunit. Dif1 is localized primarily in the cytoplasm and functions in a pathway separate from the Kap122-Wtm1 proteins. Blockage of nuclear export restores nuclear localization of R2 to the wtm1Δ but not the wtm1Δ dif1Δ cells. Dif1 is phosphorylated in response to DNA damage in a checkpoint kinase-dependent manner. Interestingly, phosphorylated Dif1 is enriched in the nucleus after DNA damage. We also show that regulation of Sml1 and Dif1 phosphorylation and proteolysis occurs through a homologous domain shared between the two proteins. Taken together, our results indicate that Dif1 plays an important role in nuclear import of the R2 subunit and works concertedly with Kap122-Wtm1 to control the dynamic RNR subcellular localization in response to genotoxic stress.