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J. Biol. Chem., Vol. 282, Issue 23, 16820-16828, June 8, 2007

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p53R2-dependent Ribonucleotide Reduction Provides Deoxyribonucleotides in Quiescent Human Fibroblasts in the Absence of Induced DNA Damage^*

Giovanna Pontarin, Paola Ferraro, Pelle Håkansson, Lars Thelander, Peter Reichard, and Vera Bianchi¹

From the Department of Biology, University of Padova, I-35131 Padova, Italy and the Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden

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 [5-³H]cytidine, [6-³H]deoxycytidine, and [C³H₃]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.

Received for publication, February 14, 2007 , and in revised form, March 27, 2007.

^* This work was supported by grants from the Italian Association for Cancer Research, Italian Telethon Grant GGP05001, the Italian Ministry of Education and Research Prin Project 2005 (to V. B.), the Swedish Research Council, the Medical Faculty of Umeå University, and the Kempe Foundation (to L. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 39-0498276282; Fax: 39-0498276280; E-mail: vbianchi{at}bio.unipd.it.

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