DNA Replication Kinetics in X-Irradiated Chinese Hamster Ovary Cells

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Abstract
The kinetics of semiconservative DNA replication were studied in both asynchronous and synchronized Chinese hamster ovary cells (CHO) irradiated with X-ray doses up to 3000 rad. Amounts of DNA replicated were determined by isopycnic gradient centrifugation of DNA from cells which were incubated after irradiation in medium containing 5-bromodeoxyuridine (50 .mu.g/ml) and 5-fluorodeoxyuridine (0.1 .mu.g/ml). Cells irradiated in early G1 phase experienced a delay in their entry into S phase. This G1 block was dose independent in the range from 300-3000 rad and was 0.5-0.7 h in length. Cells at the G1/S boundary were insensitive to X-ray-induced perturbations of bulk DNA synthetic rates when exposed to doses less than 1000 rad. At doses in excess of 1000 rad, these cells were inhibited from replicating their DNA for a time, but ultimately replicated near-control levels of their DNA. Cells irradiated in S phase again showed no effects of X-ray doses below 1000 rad on their ability to replicate bulk DNA. After a 3000 rad exposure the rate of DNA replication in these S-phase cells was markedly reduced compared to that of controls. Irradiation of asynchronous cells with doses from 150-3000 rad does reduce the rate of semiconservative DNA replication in these cultures in a dose-dependent manner. X-ray doses greater than 1000 rad reduced the rate of DNA synthesis in irradiated S-phase cells, thus prolonging the length of S phase. The combined data from asynchronous or synchronized cultures, irradiated with X-ray doses less than 1000 rad, indicated that at least a portion of the reduction in DNA replication rates in irradiated asynchronous CHO cultures was due to the X-ray-induced G1 block, which reduced the overall number of cells in S phase after irradiation.