Abstract
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.
Highlights
The synthesis of dNTPs occurs both in the cytosol and in mitochondria
Confluent Quiescent Human Lung Fibroblasts Lack Protein R2 but Contain an Increased Amount of p53R2—Ribonucleotide reductase activity requires the presence of R1 together with either R2 or p53R2
As a background to our kinetic isotope experiments concerning dNTP synthesis in human fibroblasts, we first analyzed by Western blotting the relative amounts of the three proteins in these cells
Summary
The synthesis of dNTPs occurs both in the cytosol and in mitochondria. In the cytosol a de novo pathway starting from small molecules involves the enzymes ribonucleotide reductase [1], dCMP deaminase [2], and thymidylate synthase [2] as the major players. In cultured cells kinetic experiments with labeled thymidine supported the existence of the two pathways for the synthesis of mt dNTPs and the predominance of the salvage pathway in quiescent cells [13] It was recently found [14] that quiescent mouse 3T3 fibroblasts obtained by serum starvation contained p53R2 [15, 16], a protein that can substitute for R2 to form an active enzyme together with R1, the second subunit of ribonucleotide reductase [17]. In these cells hydroxyurea, an inhibitor of both R2 and p53R2 [1, 17], decreased the size of dNTP pools suggesting that p53R2 was reducing ribonucleotides.
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