Effects of Thymidine on Deoxyribonucleoside Triphosphate Pools and Deoxyribonucleic Acid Synthesis in Chinese Hamster Ovary Cells

Abstract

Abstract Cultured Chinese hamster ovary cells were synchronized by isoleucine starvation. Thymidine was added either during G1 or during S-phase and deoxyribonucleotide pools and the rate of DNA synthesis were measured. Addition of 1 mm thymidine to G1 cells did not appreciably influence the entry of the cells into S-phase but inhibited the rate of DNA synthesis up to 90%. The pools of dTTP, dGTP, and dATP increased about 25-, 10-, and 2-fold, respectively, while the dCTP pool decreased to about 10% of the controls. Inhibition of DNA synthesis was completely prevented when 5 µm deoxycytidine was added together with thymidine. The dCTP pool was then almost of normal size while the other three pools were expanded. Addition of 1 mm thymidine to cells in S-phase gave similar changes in pool sizes and also inhibited DNA synthesis. The decline in the rate of DNA synthesis was correlated in time with the decrease of the dCTP pool. Addition of 5 µm deoxycytidine to thymidine-inhibited cells normalized both DNA synthesis and the dCTP pool without affecting the other three pools. These results, as well as earlier data concerning pool sizes of deoxynucleoside triphosphates in synchronized cell populations, suggest the possibility that the size of the dCTP pool may have a regulatory function for the rate of DNA synthesis. Moreover, our present results show that allosteric mechanisms shown with a purified enzyme (ribonucleoside diphosphate reductase) operate in intact cells.