The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. Here, we demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin-dependent and SUMO-dependent DSB recognition, revealing that RNF4 synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin conjugating enzymes including UBC13 and UBCH5. OTUB1 binds to E2~Ub thioester intermediates and prevent ubiquitin transfer, thereby non-catalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin, and that this stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are ϳ96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralogselective modification.Post-translational protein modifications play essential roles in regulating all aspects of cell function. Small ubiquitin-related modifiers (SUMOs) 2 are unusual post-translational modifications, because they themselves are proteins of ϳ100 amino acids (1, 2). Through covalent attachment to lysine residues in target proteins, sumoylation regulates a wide range of processes, including transcription activation, DNA synthesis and repair, nucleocytoplasmic transport, and chromosome segregation. The molecular mechanisms by which sumoylation affects individual proteins, and thus this diversity of processes, are in many cases target protein-specific. An emerging theme, however, is that sumoylation often promotes interactions between modified proteins and downstream factors containing SUMO-interacting motifs (SIMs) (1). To date, a single conserved SIM has been identified that consists of a hydrophobic core ((V/I)X(V/I)(V/I)) followed or preceded by a negatively charged cluster of amino acids (3-6). Although only a limited number of SIM-containing proteins has been functionally characterized to date, a large number of proteins contains this motif and is thus predicted to interact non-covalently with SUMO.Invertebrate organisms express only a single SUMO, whereas vertebrates express three paralogs capable of covalent conjugation to other proteins: SUMO-1, SUMO-2, and SUMO-3. SUMO-2 and SUMO-3 are ϳ96% identical to each other (and thus referred to collectively as SUMO-2/3); however, they are only ϳ45% identical to SUMO-1. Increasingly, evidence suggests that SUMO-1 and SUMO-2/3 have distinct cellular functions. Proteomic studies have, for example, shown that SUMO-1 and SUMO-2/3 are conjugated to only partially overlapping subsets of proteins (7,8). In addition, localization studies indicate that SUMO-1 and SUMO-2/3 are conjugated to unique subsets of proteins that localize to different subcellular domains (9, 10)...