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Volume 272, Number 26, Issue of June 27, 1997 pp. 16118-16124
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell Cycle-dependent Metabolism of Pyrimidine Deoxynucleoside Triphosphates in CEM Cells

(Received for publication, November 26, 1996, and in revised form, March 17, 1997)

Vera Bianchi ^§ , Stefania Borella , Chiara Rampazzo ^§ , Paola Ferraro , Francesca Calderazzo ^¶ , Luigi Chieco Bianchi ^¶ , Sven Skog and Peter Reichard ^§

From the  Department of Biology, University of Padova, Via Trieste 75, 35121 Padova, Italy, ^¶ Department of Oncology and Surgical Services, Oncology Section, Via Gattamelata 64, 35128 Padova, Italy, the  Department of Medical Radiobiology, and the ^§ Department of Medical Biochemistry and Biophysics, Medical Nobel Institute, Karolinska Institutet, 17177 Stockholm, Sweden

We incorporated ³H-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G₁ and S phase cells were separated by centrifugal elutriation, and the size and specific activity of dNTP pools were determined to study the cell cycle-dependent regulation of specific dNTP synthesizing enzymes in their metabolic context. With [³H]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivity from [5-³H]deoxycytidine into dCTP occurred almost exclusively in G₁ cells. During S phase, de novo synthesis by ribonucleotide reductase was switched on, resulting in a 70-fold dilution of [³H]dCTP, confirming that ribonucleotide reductase is an S phase-specific enzyme, whereas deoxycytidine kinase is not. [5-³H]Cytidine appeared in dCTP almost to the same extent in G₁ as in S phase, despite the S phase specificity of ribonucleotide reductase. During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding increase in ribonucleotide reductase activity. During G₁, the enzyme maintained activity to provide dNTPs for DNA repair and mitochondrial DNA synthesis. The poor incorporation of isotope from deoxycytidine into DNA earlier led to the suggestion that the nucleoside is used only for DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem. 270, 631-637). The poor phosphorylation of deoxycytidine in S phase provides a better explanation.

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