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.