Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.