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 m circle plasmid (2 m). Here we show that accumulation of 2 m in the SUMO pathway mutants siz1⌬ siz2⌬, slx5⌬, and slx8⌬ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 m. Characterization of this species from siz1⌬ siz2⌬ showed that it contains tandem copies of the 2 m sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 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 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°strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 m amplification. INTRODUCTIONThe 2 m circle is a naturally occurring 6318-base pair plasmid (2 m) present in ϳ40 -60 copies in virtually all strains of Saccharomyces (Broach and Volkert, 1991). 2 m's only known activity is self-propagation, which is accomplished via two distinct plasmid maintenance mechanisms (Jayaram et al., 2004). One of these allows the plasmid to be amplified if its copy number drops and is mediated by the plasmidencoded Flp recombinase and two DNA target sites for Flp (FRT) that are present on opposite sides of the plasmid. Flp catalyzes an intramolecular recombination reaction between the two FRT sites, thereby inverting one side of the 2 m with respect to the other. Futcher (1986) has proposed a model that explains how this activity could allow multiple copies of 2 m to be made from a single origin firing: if recombination occurs during replication between the converging replication forks (RFs), it would generate a double rolling circle where both forks chase each other around the template in the same direction. The 2 m sequence would be rereplicated until a second Flp-dependent recombination event permits convergence of the RFs.Native levels of 2 m have little effect on the growth rate of wild-type (wt) yeast (Broach and Volkert, 1991). However, in several mutants defective in the SUMO (Smt3) pathway, accumulation of high copy numbers of 2 m is associated with slow, cold-sensitive growth, and cell cycle delays caused by activation of the DNA damage checkpoint (Zhao et al., 2004;Chen et al., 2005;Dobson et al., 2005;Burgess et al., 2007;Ii et al., 2007b). These strains are also heterogeneous and form irregularly shaped colonies, presumably because different lineages contain different levels of 2 m, leading to different degrees of growth inhibition. Removal of 2 m from these strai...

Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

“…In addition, ulp2⌬ cells accumulate poly-SUMO conjugates that are responsible for some of these ulp2⌬ phenotypes (6). Consistent with the cellular role of Ulp2 in processing poly-SUMO conjugates, in vitro assays indicate that Ulp2 and its human homologs SENP6/7 are more active on poly-SUMO chains than on mono-SUMO conjugates (6,26,27,32).Sumoylation is known to control pathways of DNA replication and genome stability (4,28,45,56). The RecQ helicases are central regulators of genome stability and, like human BLM and Schizosaccharomyces pombe Rqh1, budding yeast Sgs1 is sumoylated (4,11,41).…”

“…Indeed, modification by SUMO is so common that the number of proteins targeted for sumoylation in Saccharomyces cerevisiae has been estimated to be over 500 (28). Genetic studies of budding yeast have revealed a role for sumoylation in recombination-mediated DNA repair, although the role of SUMO in this process is not completely understood (4,5,16,56).Similar to ubiquitination, the conjugation of SUMO (Smt3 in budding yeast) to substrate proteins requires an ATP-dependent E1-activating enzyme (Aos1/Uba2), an E2-conjugating enzyme (Ubc9), and one of several E3 ligases (e.g., Siz1 or Siz2). The product of this reaction is an isopeptide linkage between the C-terminal glycine residue of mature SUMO and the ε-amino group of lysine residues in target proteins.…”

“…Loss of ULP2 is tolerated but results in a number of mutant phenotypes. These include heat-sensitive growth, a nibbled colony phenotype due to 2m circle overreplication, defective meiosis, and sensitivity to DNA damage from treatment with methyl methanesulfonate (MMS) or hydroxyurea (HU) (2,6,26,44,46,56). In addition, ulp2⌬ cells accumulate poly-SUMO conjugates that are responsible for some of these ulp2⌬ phenotypes (6).…”

“…Indeed, modification by SUMO is so common that the number of proteins targeted for sumoylation in Saccharomyces cerevisiae has been estimated to be over 500 (28). Genetic studies of budding yeast have revealed a role for sumoylation in recombination-mediated DNA repair, although the role of SUMO in this process is not completely understood (4,5,16,56).…”

Genetic Evidence That Polysumoylation Bypasses the Need for a SUMO-Targeted Ub Ligase

“…Cells lacking ULP2 display heat-sensitive growth, a nibbled colony phenotype due to a 2-mm circle overreplication, a severe sporulation defect, and sensitivity to DNA damage resulting from treatment with methyl methanesulfonate (MMS) or hydroxyurea (HU) (Li and Hochstrasser 2000;Schwienhorst et al 2000;Strunnikov et al 2001;Bachant et al 2002;Bylebyl et al 2003;Chen et al 2005;Dobson et al 2005;Xiong et al 2009). Characterization of this DNA damage sensitivity revealed a unique role for Ulp2 in resuming growth following checkpoint arrest at mitosis (Schwartz et al 2007).…”