Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.DNA precursors ͉ immunofluorescence ͉ mitochondrial DNA ͉ p53R2 ͉ subcellular localization D NA replication and repair require a balanced supply of the four common deoxynucleoside triphosphates (dNTPs). In mammalian cells DNA synthesis occurs in two separate compartments: nucleus and mitochondria. The complete nuclear DNA is replicated only in cycling cells during S-phase, whereas cycling and quiescent cells replicate mitochondrial DNA and repair damaged DNA during their whole existence. Thus cycling cells require during a limited period a large supply of dNTPs in the nucleus. Outside S-phase cells consume much smaller amounts of dNTPs, mainly in the cytosol for mitochondrial (mt) DNA replication. In all cells the major supply of dNTPs comes from the de novo reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates by the enzyme ribonucleotide reductase (RNR) (1).In cycling cells, the dominant form of mammalian RNR consists of two proteins called R1 and R2. The activity of the R1/R2 enzyme is exquisitely regulated by allosteric mechanisms involving nucleoside triphosphates and also by S-phase-specific transcription and proteasome-mediated degradation of R2 in late mitosis (2). Thus postmitotic cells are completely devoid of protein R2. How do these cells synthesize dNTPs for mitochondrial DNA replication and DNA repair? Until recently the answer to this question was by salvage of deoxynucleosides but the picture changed su...

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...

African sleeping sickness is a fatal disease caused by two parasite subspecies: Trypanosoma brucei gambiense and T. b. rhodesiense. We previously reported that trypanosomes have extraordinary low CTP pools compared with mammalian cells. Trypanosomes also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. In this study, we have expressed and purified recombinant T. brucei CTP synthetase. The enzyme has a higher K m value for UTP than the mammalian CTP synthetase, which in combination with a lower UTP pool may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase is irreversibly inhibited by the glutamine analogue acivicin, a drug extensively tested as an antitumor agent. There is a rapid uptake of acivicin in mice both given intraperitoneally and orally by gavage. Daily injection of acivicin in trypanosome-infected mice suppressed the infection up to one month without any significant loss of weight. Experiments with cultured bloodstream T. brucei showed that acivicin is trypanocidal if present at 1 M concentration for at least 4 days. Therefore, acivicin may qualify as a drug with "desirable" properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi.African sleeping sickness is caused by a unicellular protozoan belonging to the class of zooflagellates (1). Two subspecies are responsible for causing the sickness in humans: Trypanosoma brucei gambiense and T. b. rhodesiense. The former one is affecting countries in western and central Africa, causing a chronic form of the sickness where the symptoms will not be evident until months or even years after the infection. T. b. rhodesiense on the other hand, is restricted to eastern and southern Africa, causing an acute illness within a few weeks after the infection (2). The trypanosomes are spread by tsetse flies and transmitted to humans in fly saliva upon a bite. A variant specific glycoprotein (VSG) coat enables the parasite to escape destruction by the host immune system (3, 4) and to eventually cause a fatal invasion of the central nervous system. There are only two drugs known to be effective against the late stage of the disease, DL-␣-difluoromethylornithine (DFMO, 2 eflornithine) and Melarsoprol. DFMO can only cure T. b. gambiense infections. Furthermore, because of the lengthy infusion schedules, it can only be administrated in a hospital setting. Melarsoprol, an old arsenical derivative, also has to be given by infusion but in addition is causing serious side effects such as fatal encephalopathy in as high as 10% of the cases. Furthermore, there is an increasing resistance to Melarsoprol reaching almost 30% in central Africa (2). Because of the highly variable nature of the glycoprotein coat, all attempts to develop an efficient vaccine have met with little success.CTP pools in trypanosomes were previously demonstrated to be very low compared with other eukaryotic cells and trypanosomes totally lack the ability t...