Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

Tatsuya,; Mullen, Janet R.; Slagle, Christopher E.; Brill, Steven J.

doi:10.1016/j.dnarep.2007.06.004

Cited by 48 publications

(53 citation statements)

References 60 publications

(82 reference statements)

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…The natural substrates of Slx5⅐Slx8-dependent ubiquitylation are unknown. The siz1⌬ siz2⌬ double mutant, slx5⌬, slx8⌬, ulp1, and certain NPC mutants accumulate as much as 10-fold higher than wt levels of 2 m DNA (Zhao et al, 2004;Chen et al, 2005;Dobson et al, 2005;Burgess et al, 2007;Ii et al, 2007b). A potential mechanism for SUMO's role in 2 m stability is suggested by the fact that Flp recombinase is modified by SUMO at a fairly high level (ϳ10%; Chen et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

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

Xiong

Silver

Ahmed

et al. 2009

MBoC

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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...

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Xiong

Silver

Ahmed

et al. 2009

MBoC

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“…As shown in Figure 7B, the slx5D strain could not survive in the presence of excess ULP2 activity. sgs1D slx5D synthetic lethality cannot be due simply to the accumulation of bulk poly-SUMO chains: One of the consequences of the loss of SLX5 is that sumoylated and polysumoylated proteins accumulate in vivo (Wang et al 2006;Ii et al 2007;Uzunova et al 2007;Wang and Prelich 2009). Deletion of SGS1 has also been associated with an increase in poly-SUMO levels (Mullen and Brill 2008).…”

Section: Resultsmentioning

confidence: 99%

“…Both ulp2D and slx5D/ slx8D mutants accumulate high levels of sumoylated proteins and poly-SUMO chains (Li and Hochstrasser 2000;Bylebyl et al 2003;Wang et al 2006;Ii et al 2007;Uzunova et al 2007) through mechanisms that are thought to be well understood. Ulp2 is best known for reducing or eliminating poly-SUMO chains, as opposed to removing mono-SUMO moieties from target proteins (Mukhopadhyay et al 2006;Mukhopadhyay and Dasso 2007;Lima and Reverter 2008).…”

Section: Discussionmentioning

confidence: 99%

“…Unlike ulp2D, these mutants are not sensitive to continuous exposure to MMS (Mullen et al 2001). Importantly, slx5D and slx8D cells, like their corresponding mutants in Schizosaccharomyces pombe, display an increase in poly-SUMO chains, which is reminiscent of that observed in the absence of ULP2 (Wang et al 2006;Ii et al 2007;Kosoy et al 2007;Prudden et al 2007;Sun et al 2007;Uzunova et al 2007). In this case, however, SUMO chains accumulate due to a decrease in proteasomal-dependent degradation as opposed to the loss of the SUMO editing function of Ulp2.…”

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confidence: 94%

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

2011

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Saccharomyces cerevisiae cells lacking the Slx5-Slx8 SUMO-targeted Ub ligase display increased levels of sumoylated and polysumoylated proteins, and they are inviable in the absence of the Sgs1 DNA helicase. One explanation for this inviability is that one or more sumoylated proteins accumulate to toxic levels in sgs1D slx5D cells. To address this possibility, we isolated a second-site suppressor of sgs1D slx5D synthetic lethality and identified it as an allele of the ULP2 SUMO isopeptidase. The suppressor, ulp2-D623H, behaved like the ulp2D allele in its sensitivity to heat, DNA replication stress, and DNA damage. Surprisingly, deletion of ULP2, which is known to promote the accumulation of poly-SUMO chains, suppressed sgs1D slx5D synthetic lethality and the slx5D sporulation defect. Further, ulp2D's growth sensitivities were found to be suppressed in ulp2D slx5D double mutants. This mutual suppression indicates that SLX5-SLX8 and ULP2 interact antagonistically. However, the suppressed strain sgs1D slx5D ulp2-D623H displayed even higher levels of sumoylated proteins than the corresponding double mutants. Thus, sgs1D slx5D synthetic lethality cannot be due simply to high levels of bulk sumoylated proteins. We speculate that the loss of ULP2 suppresses the toxicity of the sumoylated proteins that accumulate in slx5D-slx8D cells by permitting the extension of poly-SUMO chains on specific target proteins. This additional modification might attenuate the activity of the target proteins or channel them into alternative pathways for proteolytic degradation. In support of this latter possibility we find that the WSS1 isopeptidase is required for suppression by ulp2D.

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Slx5p‐Slx8p Promotes Accurate Chromosome Segregation by Mediating the Degradation of Synaptonemal Complex Components during Meiosis

et al. 2020

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Meiosis increases genetic diversity, yet the genome complement needs to be stable to ensure offspring viability. Both small ubiquitin‐like modifier (SUMO) and ubiquitin have been reported to participate in meiotic regulation, yet functions of the SUMO‐ubiquitination crosstalk in meiosis remain unclear. Here, it is reported that a SUMO‐targeted ubiquitin ligase, Slx8p, promotes accurate chromosome segregation during meiosis, since the deletion of SLX8 leads to increased aneuploidy due to a defect in synaptonemal complex (SC) component degradation. Both the RING domain and SUMO interacting motifs of Slx8p are essential for meiotic progression and maintaining spore viability, and the expression of tetraubiquitin fused with SUMO partially rescues meiotic defects in the SLX8‐deletion strain. Furthermore, Slx5p‐Slx8p can directly add ubiquitin to SUMOylated Zip1p and Ecm11p, and forced degradation of Ecm11p partially rescues the sporulation defects of the SLX8 deletion strain. These findings provide a mechanism for SC disassembly and reveal that the crosstalk between SUMOylation and ubiquitination facilitates accurate chromosome segregation by promoting SC component degradation during meiosis.

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Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

Cited by 48 publications

References 60 publications

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

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

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

Slx5p‐Slx8p Promotes Accurate Chromosome Segregation by Mediating the Degradation of Synaptonemal Complex Components during Meiosis

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