Microinjection of deoxynucleotides into mouse cells. No evidence that precursors for DNA synthesis are channeled.

The technique of microinjection was applied for the introduction of radioactive, phosphorylated precursors of DNA synthesis into living mouse cells in culture. Autoradiographs proved that DNA was well labeled by injected dTTP, dCTP, dATP, dGTP, and (to a minor extent) dTMP, but the efficiency was much lower when CDP, ADP, or GDP was used. For practical reasons, injections into nuclei were preferred, but injections into cytoplasm showed no principal difference with the autoradiographed nuclei. The kinetics of the uptake of the injected material agreed neatly with the calculation (from pool sizes and polymerization rate) that the intracellular deoxytriphosphates are sufficient for about 10 min of DNA synthesis. All this evidence strongly argues against the concept that precursors of DNA synthesis are channeled in vivo within a multienzyme complex and suggests a free diffusion of deoxynucleotides within the cell. Injected thymidine was less able to enter the deoxy nucleotide metabolism compared with thymidine from the culture medium. A mutant cell line deficient in thymidine kinase did not accumulate intracellular thymidine. These data indicate that thymidine kinase is a membrane associated enzyme and that uptake and phosphorylation of thymidine are coupled reactions.