Two-stage incorporation of thymidine triphosphate into mammalian DNA as indicated by chromatography on benzoylated DEAE-cellulose.

Replicating DNA contains single-stranded regions as indicated by the extent of its recovery in the final fraction, following stepwise elution of sheared preparations from benzoylated DEAE-cellulose using 0.3 M sodium chloride, 1.0 M sodium chloride and caffeine solutions, respectively. In preparations of hepatic DNA, isolated up to 60 min after administration of [3H]thymidine to rats subjected earlier to partial hepatectomy, the proportion of radioactivity contained in the caffeine-eluted fraction progressively decreased. This change was attributable to migration of incorporated radioactivity from replicating forms to mature (double-stranded) DNA, the latter being recovered from the column in 1.0 M sodium chloride. In terms of the same chromatograms, the relative size of the 0.3 M sodium chloride-eluted fraction also decreased, this fraction of radioactivity co-eluting from benzoylated DEAE-cellulose with thymidine triphosphate. The precursor--intermediate--product relationship between the three products separated by benzoylated DEAE-cellulose chromatography was confirmed using DNA isolated from cultured mammalian cells. Early eluting radioactivity co-chromatographed with thymidine triphosphate on thin-layer cellulose, whilst the intermediate status of caffeine-eluted radioactivity was confirmed following pulse-chase labelling procedures. Utilizing stepwise chromatography of such DNA on benzoylated DEAE-cellulose, the effect of three inhibitors could thus be described as affecting either an early or late stage of DNA synthesis. Such an approach offers a simple quantitative method of monitoring influences on DNA synthesis.