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

Abstract

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 μm circle plasmid (2 μm). Here we show that accumulation of 2 μm in the SUMO pathway mutants siz1Δ siz2Δ, slx5Δ, and slx8Δ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 μm. Characterization of this species from siz1Δ siz2Δ showed that it contains tandem copies of the 2 μm sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 μm–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 μm 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° strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 μm amplification.