Oxo‐Bridged Polyiron Centers in Biology and Chemistry

Ribonucleotide reductase activity is essential for progression through the cell cycle, catalyzing the rate-limiting step for the production of deoxyribonucleotides needed for DNA synthesis. The enzymatic activity of the enzyme fluctuates in the cell cycle with an activity maximum in S phase. We have identified and characterized two Saccharomyces cerevisiae genes encoding the regulatory subunit of ribonucleotide reductase, RNR1 and RNR3. They share approximately 80% amino acid identity with each other and 60% with the mammalian homolog, M1. Genetic disruption reveals that the RNR1 gene is essential for mitotic viability, whereas the RNR3 gene is not essential. A high-copy-number clone of RNR3 is able to suppress the lethality of rnr1 mutations. Analysis of mRNA levels in cell-cycle-synchronized cultures reveals that the RNR1 mRNA is tightly cell-cycle regulated, fluctuating 15- to 30-fold, and is coordinately regulated with the POL1 mRNA, being expressed in the late G1 and S phases of the cell cycle. Progression from the alpha-factor-induced G1 block to induction of RNR1 mRNA is blocked by cycloheximide, further defining the requirement for protein synthesis in the G1- to S-phase transition. Both RNR1 and RNR3 transcripts are inducible by treatments that damage DNA, such as 4-nitroquinoline-1-oxide and methylmethanesulfonate, or block DNA replication, such as hydroxyurea. RNR1 is inducible 3- to 5-fold, and RNR3 is inducible greater than 100-fold. When MATa cells are arrested in G1 by alpha-factor, RNR1 and RNR3 mRNA is still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. Inhibition of ribonucleotide reductase activity by hydroxyurea treatment results in arrest of the cell cycle in S phase as large budded, uninucleate cells. This specific cell-cycle arrest is independent of the RAD9 gene, defining a separate pathway for the coordination of DNA synthesis and cell-cycle progression.

/pdf/two-genes-differentially-regulated-in-the-cell-cycle-and-by-1uc4zzmd52.pdf

Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase.

Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits.

Citric Acid Fermentation

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designatedGRX1 and GRX2, which share 40–52% identity and 61–76% similarity with ...

The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species.

Reductive elimination of the 2′-hydroxyl group from ribonucleotides to yield 2′-deoxyribonucleotides, the monomeric precursors of DNA, requires an uncommon type of enzyme catalysis in which the transition metals, manganese, iron, or cobalt, and free radical intermediates cooperate In the group of deoxyadenosylcobalamin (coenzyme B 12)-dependent ribonucleotide reductases the coenzyme supplies a transient radical pair of deoxyadenosyl· and cob(II)alamin whereas in the nonheme-iron group of enzymes a protein subunit carries a stable tyrosyl radical coordinated to a binuclear iron(III) complex; the manganese-dependent enzymes are less precisely known The radicals are thought to function in hydrogen transfer from cysteine SH to the ribonucleotide substrate, and the metal complexes are apparently needed to generate and stabilize the radicals In aerobic organisms oxygen also plays a critical role in these processes and hence in DNA synthesis and cell proliferation In addition to the transition metals, Mg2+ or Ca2+ are required by several ribonucleotide reductases for structural integrity However the most potent inhibitors of deoxy-ribonucleotide biosynthesis (of potential interest in chemotherapy) are not metal chelators but radical scavengers Cell cycle arrest and cell death produced by simple chemicals like N-hydroxyurea and hydroxamates can be traced back to their reaction with ribonucleotide reductase Evidence is accumulating that independent enzymes of deoxyribonucleotide and DNA synthesis are functionally coupled in a novel type of supramolecular structure

The ribonucleotide reductases — A unique group of metalloenzymes essential for cell proliferation

Ribonucleotide reductase, the central enzyme of DNA precursor biosynthesis, has been isolated and characterized from baker's yeast. The enzyme activity, measured in extracts from three different, exponentially growing yeast strains, is high enough to meet the substrate requirement of DNA replication, in contrast to very low activities found in most other organisms. In thymidylate-permeable yeast cells ribonucleotide reductase activity is stimulated under both starvation and excess of intracellular dTMP. On the other hand growth of yeast in presence of 20 mM hydroxyurea did not increase enzyme activity.



Yeast ribonucleotide reductase is composed of two non-identical subunits, inactive separately, of which one binds to immobilized dATP. The relative molecular mass of the holoenzyme is about 250000. The enzyme reduces all four natural ribonucleoside diphosphates with comparable efficacy. GDP reduction requires dTTP as effector, ADP reduction is stimulated by dGTP, whereas pyrimidine nucleotide reduction is stimulated by any deoxyribonuclotide and ATP. Enzyme activity is independent of exogenous metal ions and is insensitive towards chelating agents. Hydroxyurea inactivates yeast ribonucleotide reductase in a slow reaction; half-inhibition (Iso) is reached only at 2 – 6 mM hydroxyurea concentration. Up to 50% reactivation occurs spontaneously after removal of the inhibitor. In accord with previous attempts by others, extensive purification of the yeast enzyme has failed owing to its extreme instability in solution; the half-life of about 11 h could not be influenced by any protective measure.



Taken together, yeast ribonucleotide reductase combines features known from Escherichia coli and mammalian enzymes with differing, individual properties.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1984.tb08099.x

Deoxyribonucleotide biosynthesis in yeast (Saccharomyces cerevisiae). A ribonucleotide reductase system of sufficient activity for DNA synthesis.

Deoxyribonucleotide biosynthesis in yeast: assay and properties of ribonucleotide reductase in permeabilized Saccharomyces cerevisiae cells.

Ribonucleoside diphosphate reductases isolated from Escherichia coli, baker's yeast, Ehrlich ascites tumor cells, and unicellular green alga (Scenedesmus obliquus) are inhibited strongly and uniformly by the polymeric triphenylmethane dye, aurintricarboxylic acid. The molecule appears to interact simultaneously with the enzyme's various nucleotide and catalytic (iron-organic radical) sites. Oligo- and polyribonucleotides are also inhibitory. These reactions serve as models of the probably physiologic regulation of ribonucleotide reduction exerted by natural inhibitors. Partial characterization of an inhibitor fraction found in wheat seed embryo is described.

Manfred Lammers

Papers

The ribonucleotide reductases — A unique group of metalloenzymes essential for cell proliferation

Deoxyribonucleotide biosynthesis in yeast (Saccharomyces cerevisiae). A ribonucleotide reductase system of sufficient activity for DNA synthesis.

Deoxyribonucleotide biosynthesis in yeast: assay and properties of ribonucleotide reductase in permeabilized Saccharomyces cerevisiae cells.

Aurintricarboxylic acid and polynucleotides as novel inhibitors of ribonucleotide reductases.