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...

African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavagedependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K m ‫؍‬ 0.04 -0.08 M depending on [phosphate]) and by being negatively regulated by adenosine (K i ‫؍‬ 8 -14 M). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.Trypanosoma brucei is an extracellular parasite that is transmitted by tsetse flies and lives in the blood, lymph, and central nervous system of its mammalian hosts (1, 2). The parasite causes African sleeping sickness in humans and nagana in cattle. There are two variants of African sleeping sickness, a chronic form caused by the subspecies Trypanosoma brucei gambiense and an acute form caused by Trypanosoma brucei rhodesiense. Both variants are fatal, but the chronic form has a slower progress. Current treatment is unsatisfactory because of low efficacy and high toxicity. Therefore, there is a great need of new drugs to treat the disease, especially at later stages when the parasites infect the brain. Promising results with the adenosine analog cordycepin (3Ј-deoxyadenosine) on T. brucei-infected mice with brain infection suggest that adenosine analogs can be developed into new antitrypanosomal agents (3).Unlike mammalian cells, trypanosomes lack de novo purine biosynthesis, and they are therefore totally dependent on purine salvage (4). The major purine source in human blood is a matter of controversy; when the blood was directly mixed with an adenosine deaminase inhibitor to prevent purine degradation, adenosine was present at 2 M concentration, whereas hypoxanthine (0.7 M) and inosine (0.2 M) were minor sources (5). However, other r...

SummaryUnderstanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.