CELL CYCLE-DEPENDENT METABOLISM OF PYRIMIDINE DEOXYNUCLEOSIDE TRIPHOSPHATES IN CEM CELLS

We incorporated H-3-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G(1) and S phase ce lls were separated by centrifugal elutriation, and the size and specif ic activity of dNTP pools were determined to study the cell cycle-depe ndent regulation of specific dNTP synthesizing enzymes in their metabo lic context. With [H-3]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivit y from [5-H-3]deoxycytidine into dCTP occurred almost exclusively in G (1) cells, During S phase, de novo synthesis by ribonucleotide reducta se was switched on, resulting in a 70-fold dilution of [H-3]dCTP, conf irming that ribonucleotide reductase is an S phase-specific enzyme, wh ereas deoxycytidine kinase is not, [5-H-3]Cytidine appeared in dCTP al most to the same extent in G(1) as in S phase, despite the S phase spe cificity of ribonucleotide reductase, During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding incr ease in ribonucleotide reductase activity. During G(1), the enzyme mai ntained 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.