Genetic recombination of homologous plasmids catalyzed by cell-free extracts of Saccharomyces cerevisiae.
We have developed an in vitro system utilizing yeast cell-free extracts to catalyze recombination events between homologous plasmids containing different mutant alleles of the Tet or ARG4 genes. The reaction increased the frequency of Tcr or Arg+ transformants (recombinants) from 2 X 10(-6) to 1-3 X 10(-3). Linearizing one substrate between the two tet mutations stimulated the reaction 2 to 4 fold. The reaction required rATP, Mg++, NAD, and DTT. The rad52-1 mutation decreased the reaction between linear and circular substrates 5 to 6 fold but had little effect with circular substrates. The structures of Tcr plasmids was analyzed by restriction endonuclease mapping and was consistent with a recombination reaction involving crossing-over and gene conversion. Recombination products were also observed directly by subjecting reaction mixtures to electrophoretic analysis. These results indicate that recombination events were catalyzed by the yeast extract.
D002474:Cell-Free System; D007700:Kinetics; D008938:Mitosis; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*