title: Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage.
abstract: 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.
authors.affiliation: Department of Biology, University of Padova, 35131 Padova, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Pontarin
authors.forename: Artur
authors.initials: A
authors.lastname: Fijolek
authors.forename: Paola
authors.initials: P
authors.lastname: Pizzo
authors.forename: Paola
authors.initials: P
authors.lastname: Ferraro
authors.forename: Chiara
authors.initials: C
authors.lastname: Rampazzo
authors.forename: Tullio
authors.initials: T
authors.lastname: Pozzan
authors.forename: Lars
authors.initials: L
authors.lastname: Thelander
authors.forename: Peter A
authors.initials: PA
authors.lastname: Reichard
authors.forename: Vera
authors.initials: V
authors.lastname: Bianchi
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D019556:COS Cells; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000201:enzymology; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism*; D004249:DNA Damage; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012265:Ribonucleotides; D049452:Green Fluorescent Proteins; D012264:Ribonucleotide Reductases