Cellular deoxyribonucleotide triphosphate pools levels and DNA synthesis

Abstract

Growth curves were established for mouse 3T3 cells replicating on a plastic surface and for mouse L cells in suspension culture. At different stages of growth the cells were analyzed for deoxyribonucleoside triphosphate content and in the exponential phase 3T3 cells contained pools similar to those previously found in replicating mouse embryo cells. When the 3T3 cells formed a confluent layer, the fraction of cells synthesizing DNA was very small; in such confluent cultures the pools of dCTP, dGTP and dTTP decreased while the pool of dATP remained approximately stationary. The decrease of the dCTP pool was so marked that the ratio between the dCTP and dATP pools decreased two orders of magnitude. In L cells exponentially replicating in suspension culture the pool of dATP was unusually small, while the other triphosphate pools were similar to those in other types of rapidly replicating cells. When the L cell suspensions became dense the fraction of cells synthesizing DNA decreased but the decrease was much less marked than that observed with 3T3 cells. Also, the changes of the pool levels were very limited compared to those in the 3T3 cultures. Cellular pools of the four deoxyribonucleoside triphosphates were also determined in synchronized Chinese hamster cells obtained by mitotic selection or by the addition of complete medium to isoleucine-starved cells. Using either technique the synchrony was high. Cells not involved in DNA synthesis contained significant pools of deoxyribonucleoside triphosphates, but the pools expanded when the cells proceeded through the S phase of the cell cycle. At all timepoints the dCTP pool was the largest while the dGTP pool was the smallest amounting to 1–5% of the size of the dCTP pool. The size of the dCTP pool was closely correlated to the rate of DNA synthesis while the other pools reached their highest values after DNA synthesis.