Inhibition and resumption of replicon initiation in X-irradiated mouse L5178Y cells

Cultured mouse L5178Y cells were irradiated at room temperature with various doses of X-rays, and the rate of 3H-thymidine incorporation was followed for 2 1/2h after exposure. The rate of 3H-thymidine incorporation is affected by two factors; inhibition and resumption of replicon initiation, and chain elongation. A simple method has been developed for estimating the amount of thymidine incorporation which is accounted for by DNA chain elongation after irradiation. This then allows an analysis of the inhibition and resumption of replicon initiation caused by X-rays. The method is based on knowledge of the rate of DNA chain elongation and of the distribution of replicon sizes which are determined by fiber autoradiography. The duration of the inhibition of replicon initiation and the initial slope of the curve showing recovery of initiation were obtained by subtracting the DNA synthesis due to chain elongation from the overall rate of 3H-thymidine incorporation. The interval of depressed DNA synthesis increased with increasing dose of X-rays but only gradually at doses higher than 500 rad. The interval did not exceed 60 min even after 5,000 rad. The initial shope of the curve showing recovery of initiation decreased in a dose dependent manner but very slowly at doses higher than 1,000 rad of X-rays. It appears likely that this initial slope correlates with the relative number of replicons initiating synthesis at the beginning of the recovery period.