DNA Repair and Global Sumoylation Are Regulated by Distinct Ubc9 Noncovalent Complexes

Abstract

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 ...

Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila

“…Several results from our study are consistent with data from previous investigations of Ubc9: (i) complete deletion of UBC9 was lethal, a result consistent with findings for S. cerevisiae, Caenorhabditis elegans, and mammalian cells (10,18,36,46); (ii) depletion of Ubc9p resulted in greater sensitivity to MMS and cisplatin, consistent with its role in DNA damage repair seen in other studies (41,47); and (iii) disruption of Ubc9p function resulted in defective mitosis of the micronucleus. MICs were undetectable in a large fraction of UBC9-depleted cells during vegetative growth.…”

Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels

“…Atomic-level details of Ubc9’s interaction with a few SUMO substrate proteins have been mapped in studies using X-ray crystallography and NMR [12–16]. Within target protein(s), Ubc9 recognizes a SUMOylation motif – “Ψ-K-x-D/E” – where Ψ represents a hydrophobic residue and K is the SUMO acceptor lysine [17].…”

RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway

“…STUbL E3 ligases, such as RNF4, promote the polyubiquitination and degradation of polySUMOylated proteins (35,36). RNF4 is a strong regulator of DNA damage response (19)(20)(21)(22)(41)(42)(43), suggesting that it polyubiquitinates DNA repair proteins, which are themselves polySUMOylated.…”

“…Many cellular proteins are known to undergo UBC9-mediated SUMOylation prior to their polyubiquitination and degradation by STUbL enzymes (35,36). STUbL enzymes are ubiquitin E3 ligases, which contain SIMs, and are directed to their substrates by SUMO-SIM interactions.…”