GENETIC-RECOMBINATION OF HOMOLOGOUS PLASMIDS CATALYZED BY CELL-FREE-EXTRACTS OF TOPO-ISOMERASE MUTANT STRAINS OF SACCHAROMYCES-CEREVISIAE

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used t o catalyse the recombination of bacterial plasmids in vitro. Recombina tion between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinan t plasmid DNA into Escherichia coli DH5. This in vitro recombination s ystem was used to determine the involvement of eukaryotic topo-isomera ses in genetic recombination. Cell-free extracts prepared from a tempe rature-sensitive topo-isomerase II mutant (top2-1) of S. cerevisiae yi elded tetracycline-resistant recombinants, when the recombination assa ys were performed at both a non-restrictive temperature (30-degrees-C) and the restrictive temperature (37-degrees-C). This result was obtai ned whether or not ATP was present in the recombination buffer. Extrac ts from a non-conditional topo-isomerase I mutant (top1-1) of S. cerev isiae yielded tetracycline-resistant recombinants, as did a temperatur e-sensitive double mutant (top2-1/top1-8) at the restrictive temperatu re. The results of this study indicate that neither topo-isomerase I n or topo-isomerase II was involved in the recombinational activity exam ined.