Rnr4p, a Novel Ribonucleotide Reductase Small-Subunit Protein

Abstract

Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are ␣ 2 ␤ 2 tetramers; each of the larger, ␣ subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, ␤ subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode ␣ subunits and one gene, RNR2, that encodes a ␤ subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the ␤-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Three classes of ribonucleotide reductases have been well characterized (24). Class I enzymes are found in all eukaryotes and some prokaryotes. The best-studied class I enzyme is the Escherichia coli ribonucleotide reductase (10, 30), an ␣ 2 ␤ 2 tetramer that can be decomposed to two catalytically inactive homodimers, R1 (␣ 2 ) and R2 (␤ 2 ). Each of the larger ␣ subunits possesses binding sites for substrate and allosteric effectors and also contains several redox-active cysteine residues. Each of the smaller ␤ subunits contains a binuclear Fe(III) complex. The X-ray structure of E. coli R2 reveals that the iron ions are bridged by both an O 2Ϫ ion and the carboxyl group of a glutamate residue (22). Each iron is further liganded by two carboxyl oxygen atoms from aspartate or glutamate residues, a histidine N␦ residue, and a water molecule. The recently solved structure of the mouse R2 protein indicates that the iron-binding center of eukaryotic proteins is similar to that of the E. coli protein (17). The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. The enzyme is inhibited by hydroxyurea, which specifically quenches the tyrosyl radical (19).Amino acid sequence alignments of the class 1 R2 proteins from different species identify 16 residues that are conserved in a...

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

“…This organism possesses no deoxynucleoside kinase activities, and thus, dNTP synthesis is entirely dependent on RNR (72). In S. cerevisiae, the levels of the subunits are controlled transcriptionally and the level of mRNA of the α subunit is increased during the S-phase of the cell cycle during normal growth, while the levels of mRNA for the β and β′ subunits remain largely unchanged (22,25,27,31). We have recently shown that the mRNA levels are correlated with the protein levels (D. L. Perlstein, M. Huang, and J. Stubbe, unpublished results).…”

“…In addition, a Tyr-to-Phe substitution at the Tyr designed to be oxidized has no effect on β′ function in vivo (27). Thus, ββ′ can maximally contain a single diferric-Y • cofactor located in the active site of β.…”

Ccr4 contributes to tolerance of replication stress through control ofCRT1mRNA poly(A) tail length

“…As the RNR enzyme complex is the molecular target of HU, we reasoned that genetic impairment Journal of Cell Science 119 (24) of RNR activity would hamper growth of the ccr4⌬ strain. Although RNR1 and RNR4 are essential genes in some genetic backgrounds, it is possible to delete either gene in other strain backgrounds, including in the BY4741 background used in this study; such rnr1⌬ and rnr4⌬ strains grow slowly and are hypersensitive to HU (Wang et al, 1997;Dubacq et al, 2004). We were therefore able to determine whether cells that lack Ccr4 have a genetic requirement for full RNR activity.…”

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

“…R2 houses a diferric-tyrosyl radical [(Fe) 2 -Y⅐] cofactor that is essential for catalysis. In Saccharomyces cerevisiae, there are two genes encoding R2: RNR2 (␤) and RNR4 (␤Ј) (23)(24)(25). Recent studies (26)(27)(28) have established the fact that the active form of R2 is the ␤␤Ј heterodimer (formerly designated Rnr2-Rnr4).…”