PubMed1975
From sonicated log-phase L-cells (L929) crude enzyme fractions (particulate and soluble) were prepared by differential centrifugation, and assayed for replicative DNA polymerase activity. Treatment of the cells with ara-c in small doses (0,01/mug/ml) for 4 hours (1/2 Ts) resulted in 11 percent inhibition of their DNA synthesis, while the total DNA polymerase activity was inhibited by 14 percent. However, when assayed as soluble or particulate enzyme activities, the particulate fraction (replicative DNA polymerase) was found inhibited by 42 percent while the soluble fraction (reparative DNA polymerase) was stimulated by 14 percent. The DNA synthesis of L-cells treated with ara-c, and pulse-labeled with 3H-TdR for 2, 5 or 10 minutes at 23 degrees is inhibited by 5 percent, 22 percent and 23 percent respectively. When cells labeled at this temperature are lysed on alkaline sucrose gradients and their DNA is sedimented by ultracentrifugation, the incorporation into DNA smaller than 10S (Okazaki pieces), is found to be normal or slightly inhibited. However the incorporation into high molecular weight DNA (more than 35S) is inhibited by about 50 percent. Chase experiments (at 37 degrees) after ara-c treatment and labeling at 23 degrees indicate that the decrease of labeling in high molecular weight DNA at least to some extent is caused by inability of the cells to incorporate medium sized DNA (17-20S) into this fraction, because 17-20S DNA is accumulated with prolongation of the chase. The experiments indicate that the primary action of ara-c on DNA synthesis "in vivo" in mammalian cells may be an inhibition of DNA chain polymerisation.