Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α6β2 complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

Ribonucleotide reductase (RNR) provides the cell with a balanced supply of deoxyribonucleoside triphosphates (dNTP) for DNA synthesis. In budding yeast DNA damage leads to an up-regulation of RNR activity and an increase in dNTP pools, which are essential for survival. Mammalian cells contain three non-identical subunits of RNR; that is, one homodimeric large subunit, R1, carrying the catalytic site and two variants of the homodimeric small subunit, R2 and the p53-inducible p53R2, each containing a tyrosyl free radical essential for catalysis. S-phase-specific DNA replication is supported by an RNR consisting of the R1 and R2 subunits. In contrast, DNA damage induces expression of the R1 and the p53R2 subunits. We now show that neither logarithmically growing nor G o /G 1 -synchronized mammalian cells show any major increase in their dNTP pools after DNA damage. However, non-dividing fibroblasts expressing the p53R2 protein, but not the R2 protein, have reduced dNTP levels if exposed to the RNR-specific inhibitor hydroxyurea, strongly indicating that there is ribonucleotide reduction in resting cells. The slow, 4-fold increase in p53R2 protein expression after DNA damage results in a less than 2-fold increase in the dNTP pools in G o /G 1 cells, where the pools are about 5% that of the size of the pools in S-phase cells. Our results emphasize the importance of the low constitutive levels of p53R2 in mammalian cells, which together with low levels of R1 protein may be essential for the supply of dNTPs for basal levels of DNA repair and mitochondrial DNA synthesis in G o /G 1 cells. Mammalian cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs)2 for DNA replication and repair. The rate-limiting step in the formation of DNA precursors is the de novo reduction of ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates by the enzyme ribonucleotide reductase (RNR) (1). In S phase, the mammalian RNR enzyme is composed of the homodimeric R1 and R2 subunits, which together form a heterotetrameric active enzyme. The large R1 protein (90 kDa) carries the active site, whereas the small R2 protein (45 kDa) contains a diferric iron center generating a tyrosyl free radical necessary for catalysis (1, 2). An additional mammalian RNR protein, p53R2, was identified in 2000 (3, 4). Like the homologous R2 protein, p53R2 contains a tyrosyl free radical and forms an active RNR complex with the R1 protein in vitro (5). The tyrosyl free radical of both the R2 and the p53R2 proteins is specifically destroyed by the RNR inhibitor hydroxyurea (5, 6).Because unbalanced dNTP pools can cause genetic abnormalities and cell death (1), RNR activity is tightly regulated in mammalian cells by S-phase-specific transcription of the R1 and R2 genes (7-9), binding of nucleoside triphosphate allosteric effectors to the R1 protein (10), and anaphase promoting complex-Cdh1-mediated degradation of the R2 protein in late mitosis (11,12). In cycling cells, the S-phase-dependent activity of the RNR complex is l...

Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an ␣ 2 ␤ 2 complex consisting of R1 (␣) and R2 (␤) proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/ dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (␣ 6 ␤ 2 ). The newly discovered ␣ 6 ␤ 2 complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.Ribonucleotide reductase is a key enzyme to synthesize a balanced supply of the four dNTPs used as building blocks for DNA synthesis (1). The mammalian ribonucleotide reductase reduces ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates, which are further metabolized in the cell to become dCTP, dTTP, dATP, and dGTP. This enzyme consists of two different proteins called R1 (␣) and R2 (␤) that are both required for enzymatic activity. The R2 protein is a dimer (2 ϫ 45 kDa), and each polypeptide contains a tyrosyl radical that is generated and stabilized by an iron center. Electrons are shuttled between the tyrosyl radical and the active site in the R1 protein where the actual catalysis occurs. The R1 protein contains a substrate-binding site and two allosteric effector-binding sites termed the specificity and activity sites, respectively. ATP and dATP bind to both allosteric sites, whereas dGTP and dTTP bind to only the specificity site. In the absence of nucleotide effectors, the mammalian R1 protein is a 90-kDa monomer. Allosteric effectors that only bind to the specificity site (dTTP or dGTP) induce the formation of an enzymatically active ␣ 2 ␤ 2 complex by stimulating R1 dimer formation and R1-R2 interaction (2).The specificity site determines which substrate is to be reduced. When ATP (or dATP used at low concentration) is bound to this site, the enzyme reduces CDP and UDP. In a similar manner, dTTP stimulates GDP reduction and dGTP stimulates ADP reduction. By having this regulation, the enzyme ensures that there will be a balanced supply of all four dNTPs in the cell. The mechanism behind the specificity site function is known from experiments where the R1 dimer has been crystallized together with various allosteric effectors and substrates. When a specificity site effector is bound to one of the two R1 polypeptides, a conformational change is induced in a connecting loop that influences binding of the correct substrate to the second R1 polypeptide (3). Therefore, R1 ...

Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (␣) and R2 (␤). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited ␣ 4 ␤ 4 complex in the presence of dATP and an active ␣ 2 ␤ 2 complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30 -50 times stronger in the ␣ 4 ␤ 4 complex than in the ␣ 2 ␤ 2 complex, which was in equilibrium with free ␣ 2 and ␤ 2 subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form ␣ 4 ␤ 4 complexes. ATP could also induce the formation of a generally inhibited ␣ 4 ␤ 4 complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for ␣ 4 ␤ 4 formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/ dTTP and forms active and inactive ␣ 6 ␤ 2 complexes. Ribonucleotide reductase (RNR)3 is a key enzyme for the synthesis of DNA building blocks as it converts ribonucleoside-5Ј-dior triphosphates to the corresponding deoxyribonucleotides (1). It is important that the synthesis of these building blocks is regulated to avoid an increased mutation rate caused by perturbed dNTP levels (2, 3). RNRs can be divided into three different classes (I, II, and III) mainly based on different cofactors for the catalytic activity, oxygen dependence, and mechanism for free radical generation (1). Class Ia RNRs (1) contain two non-identical subunits, denoted R1 (␣) and R2 (␤), that are both needed for enzyme activity. In bacteria, the two subunits are often referred to as NrdA and NrdB, respectively. The required radical for ribonucleotide reduction is generated on a tyrosine residue in the R2 protein and transferred via a radical transfer pathway to the R1 protein where the catalysis occurs. The R1 protein contains the substrate binding site and two allosteric sites termed the specificity and overall activity sites. Based on differences in allosteric regulation and polypeptide sequence, class I RNRs are further subgrouped into class Ia and Ib (4). Class Ia enzymes have an overall activity site, whereas class Ib lacks the N-terminal region where the overall activity site is located.Class Ia RNRs are found almost in all eukaryotic organisms and some bacteria, viruses, and bacteriophages (see the Ribonucleotide Reductase Database (RNRdb)). The first RNR studied was from...

8-Oxo-7,8-dihydroguanine- (8-oxoguanine-) containing nucleotides are generated in the cellular nucleotide pool by the action of oxygen radicals produced during normal cellular metabolism. We examined the interconversion and metabolic fate of 8-oxoguanine-containing ribonucleotides in mammalian cells. (1) 8-OxoGTP can be generated not only by direct oxidation of GTP but also by phosphorylation of 8-oxoGDP by nucleotide diphosphate kinase, and the 8-oxoGTP thus formed can serve as a substrate for RNA polymerase II to induce transcription errors. (2) MTH1 protein carrying intrinsic 8-oxo-dGTPase activity has the potential to hydrolyze 8-oxoGTP to 8-oxoGMP, thus preventing misincorporation of 8-oxoguanine into RNA. 8-OxoGMP, the degradation product, cannot be reutilized, since guanylate kinase, which has the potential to phosphorylate both GMP and dGMP, is inactive on 8-oxoGMP. (3) Ribonucleotide reductase, which catalyzes reduction of four naturally occurring ribonucleoside diphosphates, cannot convert 8-oxoguanine-containing ribonucleotide to the deoxyribonucleotide. This step appears to serve as a gatekeeper to prevent formation of mutagenic substrates for DNA synthesis from oxidized ribonucleotides.