Stimulation of in vitro sumoylation by Slx5-Slx8: evidence for a functional interaction with the SUMO pathway.

The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2micro circle from the mutants, suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Delta and slx8Delta mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5 and Slx8 complex is capable of interacting directly with the SUMO pathway.

Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex.

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structure-specific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates.

Purification of the yeast Slx5-Slx8 protein complex and characterization of its DNA-binding activity.

SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure-function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5-Slx8 may act directly on DNA to promote genome stability.

Slx5-Slx8 ubiquitin ligase targets active pools of the Yen1 nuclease to limit crossover formation.

The repair of double-stranded DNA breaks (DSBs) by homologous recombination involves the formation of branched intermediates that can lead to crossovers following nucleolytic resolution. The nucleases Mus81-Mms4 and Yen1 are tightly controlled during the cell cycle to limit the extent of crossover formation and preserve genome integrity. Here we show that Yen1 is further regulated by sumoylation and ubiquitination. In vivo, Yen1 becomes sumoylated under conditions of DNA damage by the redundant activities of Siz1 and Siz2 SUMO ligases. Yen1 is also a substrate of the Slx5-Slx8 ubiquitin ligase. Loss of Slx5-Slx8 stabilizes the sumoylated fraction, attenuates Yen1 degradation at the G1/S transition, and results in persistent localization of Yen1 in nuclear foci. Slx5-Slx8-dependent ubiquitination of Yen1 occurs mainly at K714 and mutation of this lysine increases crossover formation during DSB repair and suppresses chromosome segregation defects in a mus81∆ background.

The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10.

Mus81-Mms4 and Rad1-Rad10 are homologous structure-specific endonucleases that cleave 3' branches from distinct substrates and are required for replication fork stability and nucleotide excision repair, respectively, in the yeast Saccharomyces cerevisiae. We explored the basis of this biochemical and genetic specificity. The Mus81-Mms4 cleavage site, a nick 5 nucleotides (nt) 5' of the flap, is determined not by the branch point, like Rad1-Rad10, but by the 5' end of the DNA strand at the flap junction. As a result, the endonucleases show inverse substrate specificity; substrates lacking a 5' end within 4 nt of the flap are cleaved poorly by Mus81-Mms4 but are cleaved well by Rad1-10. Genetically, we show that both mus81 and sgs1 mutants are sensitive to camptothecin-induced DNA damage. Further, mus81 sgs1 synthetic lethality requires homologous recombination, as does suppression of mutant phenotypes by RusA expression. These data are most easily explained by a model in which the in vivo substrate of Mus81-Mms4 and Sgs1-Top3 is a 3' flap recombination intermediate downstream of replication fork collapse.

A Lysine Desert Protects a Novel Domain in the Slx5-Slx8 SUMO Targeted Ub Ligase To Maintain Sumoylation Levels in Saccharomyces cerevisiae.

Protein modification by the small ubiquitin-like modifier (SUMO) plays important roles in genome maintenance. In Saccharomyces cerevisiae, proper regulation of sumoylation is known to be essential for viability in certain DNA repair mutants. Here, we find the opposite result; proper regulation of sumoylation is lethal in certain DNA repair mutants. Yeast cells lacking the repair factors TDP1 and WSS1 are synthetically lethal due to their redundant roles in removing Top1-DNA covalent complexes (Top1ccs). A screen for suppressors of tdp1∆ wss1∆ synthetic lethality isolated mutations in genes known to control global sumoylation levels including ULP1, ULP2, SIZ2, and SLX5 The results suggest that alternative pathways of repair become available when sumoylation levels are altered. Curiously, both suppressor mutations that were isolated in the Slx5 subunit of the SUMO-targeted Ub ligase created new lysine residues. These "slx5-K" mutations localize to a 398 amino acid domain that is completely free of lysine, and they result in the auto-ubiquitination and partial proteolysis of Slx5. The decrease in Slx5-K protein leads to the accumulation of high molecular weight SUMO conjugates, and the residual Ub ligase activity is needed to suppress inviability presumably by targeting polysumoylated Top1ccs. This "lysine desert" is found in the subset of large fungal Slx5 proteins, but not its smaller orthologs such as RNF4. The lysine desert solves a problem that Ub ligases encounter when evolving novel functional domains.

Genetic evidence that polysumoylation bypasses the need for a SUMO-targeted Ub ligase.

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 sgs1Δ slx5Δ cells. To address this possibility, we isolated a second-site suppressor of sgs1Δ slx5Δ synthetic lethality and identified it as an allele of the ULP2 SUMO isopeptidase. The suppressor, ulp2-D623H, behaved like the ulp2Δ 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 sgs1Δ slx5Δ synthetic lethality and the slx5Δ sporulation defect. Further, ulp2Δ's growth sensitivities were found to be suppressed in ulp2Δ slx5Δ double mutants. This mutual suppression indicates that SLX5-SLX8 and ULP2 interact antagonistically. However, the suppressed strain sgs1Δ slx5Δ ulp2-D623H displayed even higher levels of sumoylated proteins than the corresponding double mutants. Thus, sgs1Δ slx5Δ 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 slx5Δ-slx8Δ 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 ulp2Δ.

Wss1 is a SUMO-dependent isopeptidase that interacts genetically with the Slx5-Slx8 SUMO-targeted ubiquitin ligase.

Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in this pathway because it shares phenotypes with SLX5-SLX8 mutants, including a wss1Delta sgs1Delta synthetic-fitness defect. Here we show that Wss1, a putative metalloprotease, physically binds SUMO and displays in vitro isopeptidase activity on poly-SUMO chains. Like that of SLX5, overexpression of WSS1 suppresses sgs1Delta slx5Delta lethality and the ulp1ts growth defect. Interestingly, although Wss1 is relatively inactive on ubiquitinated substrates and poly-Ub chains, it efficiently deubiquitinates a Ub-SUMO isopeptide conjugate and a Ub-SUMO fusion protein. Wss1 was further implicated in Ub metabolism on the basis of its physical association with proteasomal subunits. The results suggest that Wss1 is a SUMO-dependent isopeptidase that acts on sumoylated substrates as they undergo proteasomal degradation.

Activation of the Slx5-Slx8 ubiquitin ligase by poly-small ubiquitin-like modifier conjugates.

Protein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5-Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5-Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5-Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5-Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5-Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5-Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain.

The yeast Slx5-Slx8 DNA integrity complex displays ubiquitin ligase activity.

Genetic studies in budding yeast have previously implicated SLX5 and SLX8 in the control of genome stability and sumoylation. These genes encode RING-finger domain proteins that form a complex of unknown function. Because RING-finger proteins comprise a large class of ubiquitin (Ub) ligases, Slx5 and Slx8 were tested for this activity. Here we show that the Slx5-Slx8 complex, but not its individual subunits, stimulates several human and yeast Ub conjugating enzymes, including Ubc1, 4, 5, and Ubc13-Mms2. The RING-finger domains of both subunits are genetically required for suppression of slx sgs1Delta synthetic-lethality, and point mutations that abolish Ub ligase activity in vitro also eliminate in vivo complementation. Targets of the in vitro ubiquitination reaction include the Slx5 and Slx8 subunits themselves, and the homologous recombination proteins Rad52 and Rad57. We propose that the Slx5-Slx8 complex functions as a two-component Ub ligase in vivo and that it controls genome stability and sumoylation via ubiquitination.