Regulating the ribonucleotide reductase pathway in Saccharomyces. cerevisiae through nuclear retention and import mechanism

In response to DNA replication blocks, cells activate the D NA D amage and replication stress R esponse pathway (DDR). An important part of DDR involves the activation of the ribonucleotide reductase (RNR) pathway to generate higher levels of deoxyribonucleotide triphosphate (dNTP) for DNA replication and repair. In S. cerevisiae , the RNR enzyme complex is consist of two large subunits of Rnr1 and one of each small subunit of Rnr2 and Rnr4. One of the ways to regulate RNR activity is by sequestering Rnr2-Rnr4 in the nucleus until S-phase or during DDR, when Rnr2-Rnr4 is released from the nucleus into the cytoplasm to form active complex with the constitutively cytoplasmic Rnr1. My dissertation focuses on two S. cerevisiae genes, WTM1 ( W D-repeat containing t ranscriptional m odulator 1 ) and DIF1 (DNA D amage-regulated I mport F acilitator 1 ). Both genes are required for the proper nuclear localization of Rnr2-Rnr4. Wtm1 is a nuclear protein that physically interacts with Rnr2-Rnr4. Deletion of WTM1 leads to the loss of nuclear localization of Rnr2-Rnr4. DNA Damage or replication stress reduces the physical interaction between Wtm1 and Rnr2-Rnr4. Furthermore, forced localization of Wtm1 to the nucleolus causes Rnr2-Rnr4 to re-localize to the nucleolus. Thus, Wtm1 functions as an anchor to maintain nuclear localization of Rnr2-Rnr4 outside of the S-phase in the absence of DNA damage. Deletion of DIF1 results in the loss of nuclear Rnr2-Rnr4 similar to wtm1 Δ. Over-production of Dif1 leads to S-phase arrest in the mec1 Δ sml1 Δ mutant. Dif1 abundance is cell cycle- and DNA damage-regulated, the latter through the DDR kinase cascade, which results in the phosphorylation, inactivation, and degradation of Dif1. A dif1 Δ mutant is defective in nuclear import of Rnr2-Rnr4, in contrast to the wtm1 Δ mutant, which has functional nuclear import of Rnr2-Rnr4. Dif1 can bind to Rnr2-Rnr4 in vitro, suggesting that it is likely to be directly involved in the nuclear import of Rnr2-Rnr4. Overall, our results indicate that S. cerevisiae utilizes two separate pathways to regulate the subcellular localization of Rnr2-Rnr4: Wtm1 serves as an anchor to retain Rnr2-Rnr4 inside the nucleus, while Dif1 serves as a facilitator to import Rnr2-Rnr4 into the nucleus.