Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 mu m circle plasmid (2 mu m). Here we show that accumulation of 2 mu m in the SUMO pathway mutants siz1 Delta siz2 Delta, slx5 Delta, and slx8 Delta is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 mu m. Characterization of this species from siz1 Delta siz2 Delta showed that it contains tandem copies of the 2 mu m sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 mu m-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 mu m 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 degrees strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 mu m amplification.