Deoxyribonucleotide Pools and Deoxyribonucleic Acid Synthesis in Cultured Mouse Embryo Cells

Abstract

Abstract Cellular pools of dATP and dTTP were determined during the growth of secondary cultures of mouse embryo cells with a recently developed, highly sensitive enzymatic method. During the most active phase of DNA synthesis, the cells contained approximately 2.5 pmoles of dATP and 3.0 pmoles of dTTP per µg of DNA. Later, during growth, these values decreased to between one-half and one-third. The pools of the two nucleotides were calculated to suffice for about 2 to 4 min of DNA synthesis. After 3 days of incubation in a medium containing limiting (0.5%) serum, the cells attained a low resting level of DNA synthesis, and the pool sizes of both nucleotides dropped to around 0.5 pmole per µg of DNA. A wave of DNA synthesis was triggered after a well defined lag phase, either by raising the serum concentration of the medium to 10% or by infecting the cells with polyoma virus. With both procedures, the pools of dATP and dTTP, which increased in parallel with the rate of DNA synthesis, reached a maximum either simultaneously with or just after the peak of DNA synthesis. In no case was there a demonstrable increase in pool sizes before the onset of DNA synthesis; in fact, in the case of the dTTP pool, a decrease was observed. After serum stimulation, the activities of ribonucleotide reductase and thymidine kinase increased about 7- and 100-fold, respectively. These shifts in enzyme activities, as well as changes in pool size of deoxyribonucleoside triphosphates, demonstrate a striking parallelism between the stimulatory effects of serum and polyoma virus infection on the initiation of DNA synthesis in mouse embryo cellS.