Deoxyribonucleotide metabolism in hydroxyurea-resistant V79 hamster cells

V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20-fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonucleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5'-phosphates. The in situ activity of the reductase was only moderately elevated (1.3-fold in V79/H4 cells). In the fully resistant line, the steady-state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.