EFFECTS OF DNA TOPOISOMERASE INHIBITORS ON NONHOMOLOGOUS AND HOMOLOGOUS RECOMBINATION IN MAMMALIAN-CELLS

To study the involvement of DNA topoisomerases in recombination in mam malian cells, we used gene transfer assays to examine the effects of D NA topoisomerase inhibitors on nonhomologous (illegitimate) and homolo gous recombination. The assays were pet-formed by transfecting adenine phosphoribosyltransferase-deficient (APRT(-)) CHO cells with plasmids carrying the wild-type or mutant apri genes and by treating the cells with the inhibitors, followed by subsequent cultivation to select for APRT-positive(APRT(+))colonies. Treatments with DNA topoisomerase II inhibitors such as VP-16, VM-26, ICRF-193 resulted in a 3- to 5-fold s timulation of integration of both closed-circular and linearized plasm ids carrying the wild-type aprt gene into the recipient genome through nonhomologous recombination. The same treatments also increased 6- to 9-fold and 3-fold the number of APRT(+) recombinant colonies that wer e generated by cotransfecting two closed-circular plasmids with nonove rlapping defective aprt genes and their linearized equivalents, respec tively. However, this cotransfection assay involved intrinsically nonh omologous recombination processes; normalization of the frequencies by dividing them with those of the above nonhomologous recombination rev ealed 2-fold enhancement of homologous recombination events between th r circular mutant genes but not between the linear ones. In contrast, DNA topoisomerase I inhibitor, camptothecin, showed no such effect on tither recombination. From these results, we discuss the function of D NA topoisomerases on recombination in mammalian cells.