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.

Background: SUMO-targeted ubiquitylation controls critical cellular processes, including genome stability; but effectors and mechanisms remain undefined. Results: The Cdc48-Ufd1-Npl4 segregase binds SUMO and cooperates with the SUMO-targeted ubiquitin ligase (STUbL) in DNA repair. Conclusion: Cdc48-Ufd1-Npl4 acts as a STUbL effector. Significance: Novel dual recognition of SUMO and ubiquitin co-modified proteins likely provides selectivity and specificity in signaling by these critical factors.

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways.Conjugation of the small ubiquitin-like modifier (SUMO) to target proteins regulates many diverse processes related to genome stability and cellular growth (18-20, 31, 32, 37). SUMO is covalently attached to target proteins by a cascade that includes an E1 activating enzyme complex, a single E2 conjugating enzyme, and a limited number of E3 ligases (20). The ubiquitin modification system has a similar enzymatic cascade, but in stark contrast to the SUMO pathway, it has multiple E2s and numerous E3 ligases that provide a clear basis for selectivity (20). For example, in the fission yeast Schizosaccharomyces pombe the SUMO pathway includes a single E2 called Ubc9 (Hus5) and two known SUMO E3 ligases, Pli1 and Nse2 (51). Although these two E3 ligases are responsible for sumoylating largely distinct targets, how substrate specificity is generated is poorly characterized.Division of labor between Pli1 and Nse2 is underscored by the disparate phenotypes of cells lacking either ligase. Cells lacking Pli1 exhibit greatly reduced levels of global SUMO conjugates, heterochromatin silencing defects, and altered telomere length but are insensitive to genotoxins (38, 51, 56). Conversely, Nse2 SUMO E3 ligase-deficient cells lack the major Pli1 mutant phenotypes and are hypersensitive to genotoxic stress (51). Nse2 (Mms21 of the budding yeast Saccharomyces cerevisiae) is part of the essential Smc5/6 complex that plays critical roles in DNA repair and suppressing aberrant recombination (3,6,11,12,14,36). A phenotypic consequence of Smc5/6, Nse2/Mms21, or Ubc9 dysfunction is the accumulation of unresolved toxic recombination-dependent structures at damaged replication forks (6, 12).Interestingly, an additional factor called Rad60 (budding yeast Esc2) that physically interacts with the Smc5/6 complex was found to coact in this suppression of aberrant recombination (5,12,27,28). Rad60 defines an ...

We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe. Tech nology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch(16), using the Saccharomyces cerevisiae HO-endonuclease and its target site, MATa. DSB repair in this context was predominantly through interchromosomal gene conversion. We found that the homologous recombination (HR) genes rhp51(+), rad22A(+), rad32(+) and the nucleotide excision repair gene rad16(+) were required for efficient interchromosomal gene conversion. Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3(+). Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55Delta background involving ku70(+) and rhp51(+). Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70Delta and lig4Delta non-homologous end joining (NHEJ) mutant backgrounds compared with wild type. Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion. These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways.

Rad60 family members contain functionally enigmatic, integral SUMO-like domains (SLDs). Intriguingly, we find that despite their divergence from SUMO, each Rad60 SLD interacts with a subset of SUMO pathway enzymes. SLD2 specifically binds the SUMO E2 conjugating enzyme (Ubc9), whereas SLD1 binds the SUMO E1 activating and E3 specificity enzymes. The molecular basis of this selectivity is revealed by our 0.97 Å crystal structure of Rad60 SLD2, which shows that apart from the conserved non-substrate SUMO:Ubc9 interface, SLD2 surface features are distinct from those of SUMO. Abrogation of the SLD2:Ubc9 FEG-motif dependent interaction results in hypersensitivity to genotoxic stress, and an increase in spontaneous aberrant replication fork-associated recombination. Our results provide a mechanistic basis for the near synonymous roles of Rad60 and SUMO in survival of genotoxic stress, and suggest unprecedented DNA damage response functions for SLDs in regulating sumoylation.

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.

SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system. We show that SUMO-specific proteases (SENPs) play a crucial role in the dynamics of SUMO chains in vivo by constant deconjugation. Preventing deSUMOylation in Schizosaccharomyces pombe results in slow growth and a sensitivity to replication stress, highlighting the biological requirement for deSUMOylation dynamics. Furthermore, we present the mechanism of SUMO chain deconjugation by SENPs, which occurs via a stochastic mechanism, resulting in cleavage anywhere within a chain. Our results offer mechanistic insights into the workings of deSUMOylating proteases and highlight their importance in the homeostasis of (poly)SUMO-modified substrates.Reversible post-translational modification of proteins by ubiquitin-like covalent modifiers is a widely utilized mechanism to alter the fate, binding partners, function, or localization of a given target protein (1). A prime example is the covalent attachment of multiple ubiquitin moieties to a lysine side-chain of a target protein (2), leading to the formation of ubiquitin chains, which have different functions depending on the lysine utilized in the internal ubiquitin linkage. Recently, small ubiquitin-like modifier (SUMO) 2 has also been shown to form chains in vitro (3, 4) and in vivo (5, 6).The SUMO conjugation pathway consists of SUMO activation, transfer, and ligation enzyme machinery analogous to the ubiquitin pathway (7-9). Somewhere upwards of 500 cellular proteins are SUMOylated in vivo (10), yet the extent of observable SUMOylation is only a snapshot due to the presence of SUMO-specific proteases, SENPs. The SUMO cycle begins and ends with specific proteolytic events: the processing of pro-SUMO and the deconjugation of SUMO from the target protein by SENPs (11). In addition to monoSUMOylation, there is now growing evidence that SUMO, like ubiquitin, forms polymeric chains. Thus, the non-covalent interaction of the SUMO conjugating enzyme, Ubc9, with SUMO has presented a possible mechanism for SUMO chain formation (4, 12) and indeed in vitro, all SUMO molecules (Smt3, the Saccharomyces cerevisiae SUMO homologue, and human SUMO1, -2, and -3) have been observed to form SUMO chains, primarily via lysine residues located near their N termini (13). In vitro the SUMOylation system is quite permissive, allowing for multiple . The initial indication that SUMO chains exist in vivo came from transfection experiments yielding multimers of SUMO2 conjugated to HDAC4 (3) and from S. cerevisiae (15). More recently, evidence for in vivo SUMO chains comes from mass-spectrometry experiments using HeLa ...

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81–Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81–Eme1 is unclear. In cells lacking Nse5–Nse6 of the Smc5–Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5–Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5–Smc6 complex in HJ resolution via Mus81–Eme1.