TRANSFORMATION-ASSOCIATED RECOMBINATION BETWEEN DIVERGED AND HOMOLOGOUS DNA REPEATS IS INDUCED BY STRAND BREAKS

Rearrangements within plasmid DNA are commonly observed during transfo rmation of eukaryotic cells. One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid. We have examined the mechanisms of transformation-associ ated recombination in the yeast Saccharomyces cerevisiae using a plasm id system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmids contain homolog ous or diverged (19%) repeats of the URA3 genes (from S. cerevisiae or S. carlsbergensis) separated by the genetically detectable ADE2 colou r marker. Recombination during transformation for covalently closed ci rcular plasmids was over 100-fold more frequent than during mitotic gr owth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or sph eroplasting yield higher frequencies than electroporation. When presen t in the repeats, unique single-strand breaks that are ligatable, as w ell as double-strand breaks, lead to high levels of recombination betw een diverged and identical repeats. The transformation-associated reco mbination between repeat DNAs is under the influence of the RAD52 and RAD1 genes.