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Nucleic Acids Research, 1988, Vol. 16, No. 12 5249-5261
© 1988

Articles

Microinjected deoxynucleotides for the study of chemical inhibition of DNA synthesis

Edgar Wawra

Institut für Molekularbiologie der Universitat Wien Wasagasse 9 1090 Wien, Austria

Received April 14, 1988. Revised May 23, 1988. Accepted May 23, 1988.

Microinjection is shown to be a useful tool for studies of chemical inhibition of DNA synthesis: inhibitor-treated cells were injected with combinations of radioactive precursors and their uptake into DNA was monitored by autoradiography.

The results obtained from inhibition by cytosinearabinoside, aphidicolin, trifluorothymidine, and fluorodeoxyuridine agreed well with the common knowledge about these drugs. Short-term (but not long-term) treatments with methotrexate were compensated by injections of thymidine-nucleotides. The effect of hydroxyurea was in part, but not fully, reversed by injection of all four deoxytriphosphates; this implies a second mechanism besides inhibition of ribonucleotide reductase. Regulation of reductase was responsible for the effect of thymidine: the enhanced dTTP caused a depletion of dCTP and dATP. Novobiocin was different from all other drugs tested, DNA polymerase or enzymes of the precursor metabolism are obviously not targets of this drug.


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