p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage.
Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-(3)H]cytidine, [6-(3)H]deoxycytidine, and [C(3)H(3)]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication.
Pontarin
Giovanna
G
Department of Biology, University of Padova, I-35131 Padova, Italy.
Ferraro
Paola
P
Håkansson
Pelle
P
Thelander
Lars
L
Reichard
Peter
P
Bianchi
Vera
V
D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000502:physiology*; D002460:Cell Line; D003844:DCMP Deaminase / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D003854:Deoxyribonucleotides / Q000378:metabolism*; D006801:Humans; D012264:Ribonucleotide Reductases / Q000502:physiology*; D013940:Thymidylate Synthase / Q000378:metabolism
D018797:Cell Cycle Proteins; D003854:Deoxyribonucleotides; C405947:RRM2B protein, human; D012264:Ribonucleotide Reductases; D013940:Thymidylate Synthase; D003844:DCMP Deaminase