Instability of dicentric plasmids in yeast (spindle-mediated breakage/heterogeneous plasmid deletions/RAD52 repair/Saccharomyces cerevisiae)

Dicentric plasmids containing either two copies of centromere 4 or one copy of centromere 4 and one copy of cen- tromere 3 in the yeast plasmid vector YRp17 were constructed in vitro and introduced into yeast cells by DNA transformation. The resulting colonies were heterogeneous for a mixed population of rearranged plasmids. The rearrangements always involved dele- tion of one or both centromere sequences originally present on the plasmid. Heterogeneity was due to the continued production of deleted plasmids from a pool of unrearranged dicentric plasmids maintained within some of the yeast cells in the colony. The RAD52 gene product is known to be required for the repair of DNA dou- ble-strand breaks in yeast. Transformation of rad52 mutant yeast cells with dicentric plasmids gave rearranged plasmids similar to those observed with RAD+ yeast cells, but the transformation fre- quency was only 5-10% compared to transformation with mono- centric plasmids. Also, the ratio of unrearranged dicentric plasmid to deleted plasmids was greatly reduced in the rad52-transformed cells. These observations are consistent with a model in which cen- tromeric DNA sequences can interact independently with the