A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.     

Disruption of SUMO-targeted ubiquitin ligases Slx5–Slx8/RNF4 alters RecQ-like helicase Sgs1/BLM localization in yeast and human cells

RecQ-like helicases are a highly conserved protein family that functions during DNA repair and, when mutated in humans, is associated with cancer and/or premature aging syndromes. The budding yeast RecQ-like helicase Sgs1 has important functions in double-strand break (DSB) repair of exogenously induced breaks, as well as those that arise endogenously, for example during DNA replication. To further investigate Sgs1's regulation, we analyzed the subcellular localization of a fluorescent fusion of Sgs1 upon DNA damage. Consistent with a role in DSB repair, Sgs1 recruitment into nuclear foci in asynchronous cultures increases after ionizing radiation (IR) and after exposure to the alkylating agent methyl methanesulfonate (MMS). Yet, despite the importance of Sgs1 in replicative damage repair and in contrast to its elevated protein levels during S-phase, we find that the number of Sgs1 foci decreases upon nucleotide pool depletion by hydroxyurea (HU) treatment and that this negative regulation depends on the intra S-phase checkpoint kinase Mec1. Importantly, we identify the SUMO-targeted ubiquitin ligase (STUbL) complex Slx5–Slx8 as a negative regulator of Sgs1 foci, both spontaneously and upon replicative damage. Slx5–Slx8 regulation of Sgs1 foci is likely conserved in eukaryotes, since expression of the mammalian Slx5–Slx8 functional homologue, RNF4, restores Sgs1 focus number in slx8 cells and furthermore, knockdown of RNF4 leads to more BLM foci in U-2 OS cells. Our results point to a model where RecQ-like helicase subcellular localization is regulated by STUbLs in response to DNA damage, presumably to prevent illegitimate recombination events.     

Stability of Rad51 recombinase and persistence of Rad51 DNA repair foci depends on post-translational modifiers,ubiquitin and SUMO

The DNA double-strand breaks are particularly deleterious, especially when an error-free repair pathway is unavailable, enforcing the error-prone recombination pathways to repair the lesion. Cells can resume the cell cycle but at the expense of decreased viability due to genome rearrangements. One of the major players involved in recombinational repair of DNA damage is Rad51 recombinase, a protein responsible for presynaptic complex formation. We previously showed that an increased level of this protein promotes the usage of illegitimate recombination. Here we show that the level of Rad51 is regulated via the ubiquitin-dependent proteolytic pathway. The ubiquitination of Rad51 depends on multiple E3 enzymes, including SUMO-targeted ubiquitin ligases. We also demonstrate that Rad51 can be modified by both ubiquitin and SUMO. Moreover, its modification with ubiquitin may lead to opposite effects: degradation dependent on Rad6, Rad18, Slx8, Dia2, and the anaphase-promoting complex, or stabilization dependent on Rsp5. We also show that post-translational modifications with SUMO and ubiquitin affect Rad51's ability to form and disassemble DNA repair foci, respectively, influencing cell cycle progression and cell viability in genotoxic stress conditions. Our data suggest the existence of a complex E3 ligases network that regulates Rad51 recombinase's turnover, its molecular activity, and access to DNA, limiting it to the proportions optimal for the actual cell cycle stage and growth conditions, e.g., stress. Dysregulation of this network would result in a drop in cell viability due to uncontrolled genome rearrangement in the yeast cells. In mammals would promote the development of genetic diseases and cancer.     

The yeast Hex3.Slx8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation

Hex3 and Slx8 are Saccharomyces cerevisiae proteins with important functions in DNA damage control and maintenance of genomic stability. Both proteins have RING domains at their C termini. Such domains are common in ubiquitin and ubiquitin-like protein ligases (E3s), but little was known about the molecular functions of either protein. In this study we identified HEX3 as a high-copy suppressor of a temperature-sensitive small ubiquitin-related modifier (SUMO) protease mutant, ulp1ts, suggesting that it may affect cellular SUMO dynamics. Remarkably, even a complete deletion of ULP1 is strongly suppressed. Hex3 forms a heterodimer with Slx8. We found that the Hex3.Slx8 complex has a robust substrate-specific E3 ubiquitin ligase activity. In this E3 complex, Slx8 appears to bear the core ligase function, with Hex3 strongly enhancing its activity. Notably, SUMO attachment to a substrate stimulates its Hex3.Slx8-dependent ubiquitination, primarily through direct noncovalent interactions between SUMO and Hex3. Our data reveal a novel mechanism of substrate targeting in which sumoylation of a protein can help trigger its subsequent ubiquitination by recruiting a SUMO-binding ubiquitin ligase.     

Nuclear organization in genome stability: SUMO connections

Recent findings show that chromatin dynamics and nuclear organization are not only important for gene regulation and DNA replication, but also for the maintenance of genome stability. In yeast, nuclear pores play a role in the maintenance of genome stability by means of the evolutionarily conserved family of SUMO-targeted Ubiquitin ligases (STUbLs). The yeast Slx5/Slx8 STUbL associates with a class of DNA breaks that are shifted to nuclear pores. Functionally Slx5/Slx8 are needed for telomere maintenance by an unusual recombination-mediated pathway. The mammalian STUbL RNF4 associates with Promyelocytic leukaemia (PML) nuclear bodies and regulates PML/PML-fusion protein stability in response to arsenic-induced stress. A subclass of PML bodies support telomere maintenance by the ALT pathway in telomerase-deficient tumors. Perturbation of nuclear organization through either loss of pore subunits in yeast, or PML body perturbation in man, can lead to gene amplifications, deletions, translocations or end-to-end telomere fusion events, thus implicating SUMO and STUbLs in the subnuclear organization of select repair events.     

SUMO-targeted ubiquitin ligases in genome stability

We identify the SUMO-Targeted Ubiquitin Ligase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO-like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N-terminus. STUbL-mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8-Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5-Slx8. Cells lacking Slx8-Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8-Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8-Rfp mutants have similar DNA repair defects.     

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.     

Acute ethanol stress induces sumoylation of conserved chromatin structural proteins in Saccharomyces cerevisiae

Stress is ubiquitous to life and can irreparably damage essential biomolecules and organelles in cells. To survive, organisms must sense and adapt to stressful conditions. One highly conserved adaptive stress response is through the posttranslational modification of proteins by the small ubiquitin-like modifier (SUMO). Here, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. We found that cells exhibit a transient sumoylation response after acute exposure to ≤7.5% vol/vol ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol exposure. Mass spectrometry analyses identified 18 proteins that are sumoylated after acute ethanol exposure, with 15 known to associate with chromatin. Upon further analysis, we found that the chromatin structural proteins Smc5 and Smc6 undergo ethanol-induced sumoylation that depends on the activity of the E3 SUMO ligase Mms21. Using cell-cycle arrest assays, we observed that Smc5 and Smc6 ethanol-induced sumoylation occurs during G1 and G2/M phases but not S phase. Acute ethanol exposure also resulted in the formation of Rad52 foci at levels comparable to Rad52 foci formation after exposure to the DNA alkylating agent methyl methanesulfonate (MMS). MMS exposure is known to induce the intra-S-phase DNA damage checkpoint via Rad53 phosphorylation, but ethanol exposure did not induce Rad53 phosphorylation. Ethanol abrogated the effect of MMS on Rad53 phosphorylation when added simultaneously. From these studies, we propose that acute ethanol exposure induces a change in chromatin leading to sumoylation of specific chromatin structural proteins.     

The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination

Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.     

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein–ubiquitin interactions.     

Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast

In mammalian and budding yeast cells treated with genotoxic agents, different proteins implicated in detecting, signalling or repairing DNA lesions form nuclear foci. We studied foci formed by proteins involved in these processes in living fission yeast cells, which is amenable to genetic and molecular analysis. Using fluorescent tags, we analysed subnuclear localisations of the DNA damage checkpoint protein Rad9, of the homologous recombination protein Rad22 and of PCNA, which are implicated in many aspects of DNA metabolism. After inducing double strand breaks (DSBs) with ionising radiations, Rad22, Rad9 and PCNA form a low number of nuclear foci. Rad9 recruitment to foci depends on the presence of Rad1, Hus1 and Rad17, but is independent of downstream checkpoint effectors and of homologous recombination proteins. Likewise, Rad22 and PCNA form foci despite inactive homologous recombination repair and impaired DNA damage checkpoint. Rad22 and Rad9 foci co-localise completely, whereas PCNA co-localises with Rad22 and Rad9 only partially. Foci do not disassemble in cells unable to repair DNA by homologous recombination. Thus, in fission yeast, DSBs are detected by the DNA damage checkpoint and are repaired by homologous recombination at a few spatially confined subnuclear compartments where Rad22, Rad9 and PCNA concentrate independently.     

DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel, SUMOylation of so-far elusive upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response. We show that HERC2 and RNF168 are novel DNA damage-dependent SUMOylation targets in human cells. In response to DSBs, both HERC2 and RNF168 were specifically modified with SUMO1 at DSB sites in a manner dependent on the SUMO E3 ligase PIAS4. SUMOylation of HERC2 was required for its DSB-induced association with RNF8 and for stabilizing the RNF8-Ubc13 complex. We also demonstrate that the ZZ Zinc finger in HERC2 defined a novel SUMO-specific binding module, which together with its concomitant SUMOylation and T4827 phosphorylation promoted binding to RNF8. Our findings provide novel insight into the regulatory complexity of how ubiquitylation and SUMOylation cooperate to orchestrate protein interactions with DSB repair foci.     

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 sgs1? 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.     

RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.     

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.     

Slx8 Removes Pli1-Dependent Protein-SUMO Conjugates Including SUMOylated Topoisomerase I to Promote Genome Stability

The SUMO-dependent ubiquitin ligase Slx8 plays key roles in promoting genome stability, including the processing of trapped Topoisomerase I (Top1) cleavage complexes and removal of toxic SUMO conjugates. We show that it is the latter function that constitutes Slx8''s primary role in fission yeast. The SUMO conjugates in question are formed by the SUMO ligase Pli1, which is necessary for limiting spontaneous homologous recombination when Top1 is present. Surprisingly there is no requirement for Pli1 to limit recombination in the vicinity of a replication fork blocked at the programmed barrier RTS1. Notably, once committed to Pli1-mediated SUMOylation Slx8 becomes essential for genotoxin resistance, limiting both spontaneous and RTS1 induced recombination, and promoting normal chromosome segregation. We show that Slx8 removes Pli1-dependent Top1-SUMO conjugates and in doing so helps to constrain recombination at RTS1. Overall our data highlight how SUMOylation and SUMO-dependent ubiquitylation by the Pli1-Slx8 axis contribute in different ways to maintain genome stability.     

Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins

The function of small ubiquitin-like modifier (SUMO)-binding proteins is key to understanding how SUMOylation regulates cellular processes. We identified two related Schizosaccharomyces pombe proteins, Rfp1 and Rfp2, each having an N-terminal SUMO-interacting motif (SIM) and a C-terminal RING-finger domain. Genetic analysis shows that Rfp1 and Rfp2 have redundant functions; together, they are essential for cell growth and genome stability. Mammalian RNF4, an active ubiquitin E3 ligase, is an orthologue of Rfp1/Rfp2. Rfp1 and Rfp2 lack E3 activity but recruit Slx8, an active RING-finger ubiquitin ligase, through a RING-RING interaction, to form a functional E3. RNF4 complements the growth and genomic stability defects of rfp1rfp2, slx8, and rfp1rfp2slx8 mutant cells. Both the Rfp-Slx8 complex and RNF4 specifically ubiquitylate artificial SUMO-containing substrates in vitro in a SUMO binding-dependent manner. SUMOylated proteins accumulate in rfp1rfp2 double-null cells, suggesting that Rfp/Slx8 proteins may promote ubiquitin-dependent degradation of SUMOylated targets. Hence, we describe a family of SIM-containing RING-finger proteins that potentially regulates eukaryotic genome stability through linking SUMO-interaction with ubiquitin conjugation.