Rnr4p, a novel ribonucleotide reductase small-subunit protein.

Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are a2b2 tetramers; each of the larger, a subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, b 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 a subunits and one gene, RNR2, that encodes a b subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the b-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 a2b2 tetramer that can be decomposed to two catalytically inactive homodimers, R1 (a2) and R2 (b2). Each of the larger a subunits possesses binding sites for substrate and allosteric effectors and also contains several redox-active cysteine residues. Each of the smaller b 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 22 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 Nd 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 all of these proteins reported to date (7, 22). Most of these conserved residues are at the iron center or close to it. Previous work has identified two genes in the yeast Saccharomyces cerevisiae (RNR1 and RNR3) that encode R1 proteins (8) and one gene (RNR2) that encodes an R2 protein (7, 13). Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the R2 proteins. However, Rnr4p lacks a number of sequence elements thought to be essential for enzymatic function. Our evidence suggests that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.