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Determination of the in vivo stoichiometry of tyrosyl radical per betabeta' in Saccharomyces cerevisiae ribonucleotide reductase.

ABSTRACT: The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y(*)) cofactor. In Saccharomyces cerevisiae, two R1s, alpha(n) and , have been identified, while R2 is a heterodimer (betabeta'). beta' cannot bind iron and generate the Y(*); consequently, the maximum amount of Y(*) per betabeta' is 1. To determine the cofactor stoichiometry in vivo, a FLAG-tagged beta ((FLAG)beta) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of (FLAG)betabeta' by immunoaffinity chromatography, which was found to have 0.45 +/- 0.08 Y(*)/(FLAG)betabeta' and a specific activity of 2.3 +/- 0.5 micromol min(-1) mg(-1). (FLAG)betabeta' isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y(*)/(FLAG)betabeta' ratio of 0.5. To determine the Y(*)/betabeta' ratio without R2 isolation, whole cell EPR and quantitative Western blots of beta were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y(*)/betabeta' ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Delta gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y(*)/betabeta' ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y(*) and modulation of Y(*) concentration is not involved in the regulation of RNR activity.

SUBMITTER: Ortigosa AD

PROVIDER: S-EPMC4674157 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Determination of the in vivo stoichiometry of tyrosyl radical per betabeta' in Saccharomyces cerevisiae ribonucleotide reductase.

Ortigosa Allison D AD Hristova Daniela D Perlstein Deborah L DL Zhang Zhen Z Huang Mingxia M Stubbe JoAnne J

Biochemistry 20061001 40

The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y(*)) cofactor. In Saccharomyces cerevisiae, two R1s, alpha(n) and , have been identified, while R2 is a heterodimer (betabeta'). beta' cannot bind iron and generate the Y(*); consequent ...[more]

PMID: 17014081

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Project description:Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1)) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1)-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053-33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.

Project description:BACKGROUND: Ty1 is a long terminal repeat retrotransposon of Saccharomyces cerevisiae, with a replication cycle similar to retrovirus replication. Structurally, Ty1 contains long terminal repeat (LTR) regions flanking the gag and pol genes that encode for the proteins that enable Ty1 mobility. Reverse transcriptase produces Ty1 complementary (c)DNA that can either be integrated back into the genome by integrase or recombined into the yeast genome through homologous recombination. The frequency of Ty1 mobility is temperature sensitive, with optimum activity occurring at 24-26°C. RESULTS: In this study, we identified two host genes that when deleted allow for high temperature Ty1 mobility: RFX1 and SML1. The protein products of these genes are both negative regulators of the enzyme ribonucleotide reductase, a key enzyme in regulating deoxyribonucleotide triphosphate (dNTP) levels in the cell. Processing of Ty1 proteins is defective at high temperature, and processing is not improved in either rfx1 or sml1 deletion strains. Ty1 mobility at high temperature is mediated by homologous recombination of Ty1 cDNA to Ty1 elements within the yeast genome. We quantified cDNA levels in wild type, rfx1 and sml1 deletion background strains at different temperatures. Southern blot analysis demonstrated that cDNA levels were not markedly different between the wild type and mutant strains as temperatures increased, indicating that the increased Ty1 mobility is not a result of increased cDNA synthesis in the mutant strains. Homologous recombination efficiency was increased in both rfx1 and sml1 deletion strains at high temperatures; the rfx1 deletion strain also had heightened homologous recombination efficiency at permissive temperatures. In the presence of the dNTP reducing agent hydroxyurea at permissive temperatures, Ty1 mobility was stimulated in the wild type and sml1 deletion strains but not in the rfx1 deletion strain. Mobility frequency was greatly reduced in all strains at high temperature. Deletion of the S-phase checkpoint pathway Dun1 kinase, which inactivates Sml1 and Rfx1, reduced Ty1 mobility at a range of temperatures. CONCLUSIONS: Levels of cellular dNTPs, as regulated by components of the S-phase checkpoint pathway, are a limiting factor in homologous recombination-mediated Ty1 mobility.

Dataset Information

Determination of the in vivo stoichiometry of tyrosyl radical per betabeta' in Saccharomyces cerevisiae ribonucleotide reductase.

Publications

Determination of the in vivo stoichiometry of tyrosyl radical per betabeta' in Saccharomyces cerevisiae ribonucleotide reductase.

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