Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductase? Toward understanding the role of Y4

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 is directly involved in the reduction, and R2 contains the diferric-tyrosyl radical (Y⅐) cofactor essential for the initiation of reduction. Saccharomyces cerevisiae has two RNRs; Y1 and Y3 correspond to R1, whereas Y2 and Y4 correspond to R2. Y4 is essential for diferric-Y⅐ formation in Y2 from apoY2, Fe 2؉ , and O2. The actual function of Y4 is controversial. Y2 and Y4 have been further characterized in an effort to understand their respective roles in nucleotide reduction. (His) 6-Y2, Y4, and (His) 6-Y4 are homodimers, isolated largely in apo form. Their CD spectra reveal that they are predominantly helical. The concentrations of Y2 and Y4 in vivo are 0. R ibonucleotide reductases (RNRs) play an essential role in DNA replication and repair by providing all of the monomeric deoxynucleotides required for these processes. The genome sequencing projects have revealed that both prokaryotes and eukaryotes possess multiple RNRs, whose functions remain to be elucidated (1). Class I RNRs are composed of two homodimeric subunits: R1 (␣2) and R2 (␤2). Both subunits are essential for activity. R1 contains the binding sites for the nucleoside diphosphate substrates and the deoxynucleotide and ATP allosteric effectors that govern which nucleotide is reduced and its rate of reduction. R2 possesses a diferric-tyrosyl radical (Y⅐) cofactor essential for initiation of nucleotide reduction on R1 (2, 3).Four genes have been identified that encode RNR subunits in Saccharomyces cerevisiae: RNR1 and RNR3 (their gene products designated Y1 and Y3) are R1 homologues, and RNR2 and RNR4 (their gene products designated Y2 and Y4) are R2 homologues (4-9). Y1 and Y3 share 80% sequence identity (4). RNR1 expression is cell cycle regulated, and the gene is essential for mitotic viability (5, 10). In contrast, RNR3 is not expressed under normal growth conditions. Transcription of RNR1 and RNR3 is inducible by DNA damage; the mRNA of the latter is up-regulated Ͼ500-fold (5). Y2 and Y4 share 56% sequence identity. Y4 contains several unusual features relative to a canonical R2. It lacks about 50 amino acid residues from the N terminus, and of the 16 amino acid residues conserved in almost all class I R2s, six have been replaced in Y4. The most notable substitutions are two histidines and a glutamate, ligands of the di-iron center, which have been replaced by two tyrosines and an arginine. Such substitutions would be expected to disrupt the ability of Y4 to bind iron. RNR2 is essential for mitotic viability (10,11). RNR4 also appears to be important for mitotic viability, but its essentiality varies with genetic background (8, 9, 12). In the one case where a Y4 knockout was shown to be lethal, the lethality could be suppressed by overexpression of Y1 and Y3 (8).The transcriptional regulation of the yeast RNR genes has been studied extensively. However, little is known about the biochem...

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

“…Secondary antibodies (Abs, horseradish peroxidase-conjugated donkey anti-rabbit Abs), Complete protease inhibitor tablets, calf intestinal alkaline phosphatase, and DNase I from bovine pancreas were obtained from Roche. His β 2 and β 2 ′ were isolated from an E. coli expression system, and the heterodimer was reconstituted as described previously (28). His ββ′ typically contained 0.1-0.2 Y • and had a specific activity of 1.5-1.8 μmol min −1 mg −1 .…”

“…Anti-FLAG agarose, 3×-FLAG peptide (three tandem copies of the FLAG epitope), Bradford reagent, and all other reagents and chemicals were obtained from Sigma-Aldrich. The concentrations of His ββ′ and FLAG ββ′ were determined using an ∊ 280-310 of 99 800 M −1 cm −1 and is reported as a concentration of monomer β or dimer ββ′ (28). Iron concentrations were determined by the ferrozine assay (34).…”

“…The Nterminus of β was chosen as the site of tag attachment, as the C-terminus is required for R1-R2 interaction (43,44). Recent studies with several different N-terminally His-tagged β constructs have demonstrated that activity is subtly dependent on the construct (15,28,45). Thus, MHY343 cells were examined for growth rate and sensitivity to genotoxic stress as a course monitor of tag effects.…”

Investigation of in Vivo Diferric Tyrosyl Radical Formation in Saccharomyces cerevisiae Rnr2 Protein

“…The active form of the RNR small subunit in budding yeast Saccharomyces cerevisiae is a heterodimer of ␤ and ␤Ј, encoded by the RNR2 and RNR4 genes, respectively (14 -16). Only ␤ is capable of iron binding and Tyr ⅐ formation, and thus there is a maximum of 1Tyr ⅐ /␤␤Ј (17)(18)(19)(20). Although ␤Ј lacks three residues required for iron binding and is unable to assemble its own metallo-cofactor (18), deletion of RNR4 causes lethality or a severe growth defect depending on the genetic backgrounds (15,16).…”

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

“…Recent studies (26)(27)(28) have established the fact that the active form of R2 is the ␤␤Ј heterodimer (formerly designated Rnr2-Rnr4). Only ␤ is capable of forming the (Fe) 2 -Y⅐ cofactor (26,27), whereas ␤Ј facilitates cofactor assembly and stabilizes the resulting holoheterodimer (26)(27)(28)(29)(30).…”