A Lysine Desert Protects a Novel Domain in the Slx5-Slx8 SUMO Targeted Ub Ligase To Maintain Sumoylation Levels in Saccharomyces cerevisiae.
ABSTRACT: 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.
Project description:Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.
Project description: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.
Project description:Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)-mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl-DNA phosphodiesterase 1 (Tdp1). This is notable, as cells lacking Tdp1 are largely aphenotypic in the vegetative cell cycle due to the existence of alternative pathways for the removal of covalent Top1-DNA adducts (Top1cc). We further identify Rad60, a SUMO mimetic and STUbL-interacting protein, and the SUMO E3 ligase Nse2 as critical Top1cc repair factors in cells lacking Tdp1. Detection of Top1ccs using chromatin immunoprecipitation and quantitative PCR shows that they are elevated in cells lacking Tdp1 and STUbL, Rad60, or Nse2 SUMO ligase activity. These unrepaired Top1ccs are shown to cause DNA damage, hyper-recombination, and checkpoint-mediated cell cycle arrest. We further determine that Tdp1 and the nucleotide excision repair endonuclease Rad16-Swi10 initiate the major Top1cc repair pathways of fission yeast. Tdp1-based repair is the predominant activity outside S phase, likely acting on transcription-coupled Top1cc. Epistasis analyses suggest that STUbL, Rad60, and Nse2 facilitate the Rad16-Swi10 pathway, parallel to Tdp1. Collectively, these results reveal a unified role for STUbL, Rad60, and Nse2 in protecting genome stability against spontaneous Top1-mediated DNA damage.
Project description:Breaking and sealing one strand of DNA is an inherent feature of chromosome metabolism to overcome torsional barriers. Failure to reseal broken DNA strands results in protein-linked DNA breaks, causing neurodegeneration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1), which removes stalled topoisomerase 1 peptides from DNA termini. Here we show that TDP1 is a substrate for modification by the small ubiquitin-like modifier SUMO. We purify SUMOylated TDP1 from mammalian cells and identify the SUMOylation site as lysine 111. While SUMOylation exhibits no impact on TDP1 catalytic activity, it promotes its accumulation at sites of DNA damage. A TDP1 SUMOylation-deficient mutant displays a reduced rate of repair of chromosomal single-strand breaks arising from transcription-associated topoisomerase 1 activity or oxidative stress. These data identify a role for SUMO during single-strand break repair, and suggest a mechanism for protecting the nervous system from genotoxic stress.
Project description: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.
Project description: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.
Project description: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.
Project description:SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.
Project description:High-resolution imaging shows that persistent DNA damage in budding yeast localizes in distinct perinuclear foci for repair. The signals that trigger DNA double-strand break (DSB) relocation or determine their destination are unknown. We show here that DSB relocation to the nuclear envelope depends on SUMOylation mediated by the E3 ligases Siz2 and Mms21. In G1, a polySUMOylation signal deposited coordinately by Mms21 and Siz2 recruits the SUMO targeted ubiquitin ligase Slx5/Slx8 to persistent breaks. Both Slx5 and Slx8 are necessary for damage relocation to nuclear pores. When targeted to an undamaged locus, however, Slx5 alone can mediate relocation in G1-phase cells, bypassing the requirement for polySUMOylation. In contrast, in S-phase cells, monoSUMOylation mediated by the Rtt107-stabilized SMC5/6-Mms21 E3 complex drives DSBs to the SUN domain protein Mps3 in a manner independent of Slx5. Slx5/Slx8 and binding to pores favor repair by ectopic break-induced replication and imprecise end-joining.
Project description:Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate-a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)-is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3' phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90?kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.