ArticleBiochemistry2006
Determination of the in vivo stoichiometry of tyrosyl radical per betabeta' in Saccharomyces cerevisiae ribonucleotide reductase.
Article in Biochemistry, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 24 citations in OpenAlex.
- In-Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy.Angewandte Chemie (International ed. in English) · 2021Article
- Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.Annual review of biochemistry · 2020Review
- Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.Biochemistry · 2020Article
- The diferric-tyrosyl radical cluster of ribonucleotide reductase and cytosolic iron-sulfur clusters have distinct and similar biogenesis requirements.The Journal of biological chemistry · 2017Article
- Choosing the right metal: case studies of class I ribonucleotide reductases.The Journal of biological chemistry · 2014Review
- Conserved electron donor complex Dre2-Tah18 is required for ribonucleotide reductase metallocofactor assembly and DNA synthesis.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Ribonucleotide reductase metallocofactor: assembly, maintenance and inhibition.Frontiers in biology · 2014Article
- Investigation of in vivo roles of the C-terminal tails of the small subunit (ββ') of Saccharomyces cerevisiae ribonucleotide reductase: contribution to cofactor formation and intersubunit association within the active holoenzyme.The Journal of biological chemistry · 2013Article
- Regulation of ribonucleotide reductase in response to iron deficiency.Molecular cell · 2011Article
- Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins.The Journal of biological chemistry · 2011Article
- Class I ribonucleotide reductases: metallocofactor assembly and repair in vitro and in vivo.Annual review of biochemistry · 2011Review
- Mechanism of inactivation of human ribonucleotide reductase with p53R2 by gemcitabine 5'-diphosphate.Biochemistry · 2009Article
- Dif1 controls subcellular localization of ribonucleotide reductase by mediating nuclear import of the R2 subunit.Molecular and cellular biology · 2008Article
- Importance of the maintenance pathway in the regulation of the activity of Escherichia coli ribonucleotide reductase.Biochemistry · 2008Article
- Enhanced subunit interactions with gemcitabine-5'-diphosphate inhibit ribonucleotide reductases.Proceedings of the National Academy of Sciences of the United States of America · 2007Article
- Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.Proceedings of the National Academy of Sciences of the United States of America · 2007Article
- A role for the yeast cell cycle/splicing factor Cdc40 in the G1/S transition.Current genetics · 2007Article
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
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.
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