The effect of x-irradiation on DNA precursor metabolism and deoxyribonucleoside triphosphate acid-soluble pools was examined. Pulse-labeling experiments indicated that the rates of incorporation into DNA of thymidine and the ribonucleosides and deoxyribonucleosides of adenine and guanine were similarly reduced in a biphasic manner with increasing doses of x-irradiation. The incorporation rate of cytidine into DNA was reduced after irradiation, while the concurrent incorporation rate of deoxycytidine was greatly stimulated. Incorporation of32 P-phosphate pulse label into nondialyzable DNA after irradiation mimicked that of thymidine; nucleotide analysis of enzymatically degraded DNA pulse-labeled with32 P-phosphate confirmed the results obtained with individual ribonucleosides and deoxyribonucleosides. The purine moieties from deoxyadenosine and deoxyguanosine were efficiently incorporated into RNA. Radiation had no detectable effect on either incorporation into RNA or on the relative distribution of radioactive label from adenosine or deoxyadenosine found as dGMP and dAMP of enzymatically degraded DNA. The dTTP, dATP, and dGTP pools were not expanded after irradiation, nor were the respective pool specific activities significantly altered. The dCTP pool alone was expanded after irradiation. The specific activity of the dCTP pool labeled with deoxycytidine was also increased and accounted for the anomalous radiation-induced stimulation of deoxycytidine incorporation into DNA. Exogenous thymidine abolished both the increased dCTP specific activity and the stimulation of deoxycytidine incorporation into DNA after irradiation but did so not by eliminating either the radiation-induced expansion of the dCTP pool size or the increased intracellular level of <tex-math>$[{}^{3}{\rm H}]\text{-}{\rm dCTP}$</tex-math> derived from <tex-math>$[{}^{3}{\rm H}]\text{-deoxycytidine}$</tex-math> but by equalizing the change in magnitude of the two parameters. A radiation effect on deoxycytidylate aminohydrolase is suggested. No radiation effect on the synthesis of dCTP from cytidine was detected. After irradiation with 800 rad, thymidine incorporation recovered to control levels, on a per DNA aliquot basis, within 4 hr. However, bulk DNA replication of irradiated cells terminated at a time not greatly different from controls (∼0.7 hr), while the DNA synthetic period, measured autoradiographically, was prolonged ∼3 hr. Thus, the S retention period after irradiation represents a time of significantly reduced DNA synthesis per S cell. The size and specific activity of the dCTP pool remained elevated at all times examined (up to 8 hr postirradiation), and radiation prevented the scheduled disappearance of the dCTP pool which normally occurs at the end of S or early G2 a result consistent with a radiation-induced lengthening of the DNA synthetic period.

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