Deficient SUMO attachment to Flp recombinase leads to homologous recombination-dependent hyperamplification of the yeast 2 microm circle plasmid

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 microm circle plasmid (2 microm). Here we show that accumulation of 2 microm in the SUMO pathway mutants siz1Delta siz2Delta, slx5Delta, and slx8Delta is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 microm. Characterization of this species from siz1Delta siz2Delta showed that it contains tandem copies of the 2 mum sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 microm-encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 microm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir(o) strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 microm amplification.