Fig. 3: Ubl-tools mediated access to heterotypically linked Ubl architectures.

a Left: Schematic representation of the two-step assembly of a heterotypically linked triUb using Srt2A and Srt5M. Right: Generation of K48/K6-linked triUb. SDS-PAGE shows Srt5M-catalyzed transpeptidation between K48-diUb and Ub-K6GGK resulting in the formation of heterotypically K48/K6-linked triUb (K48/K6-triUb). LC-MS analysis confirmed the identity of the K48/K6-triUb. Full gels and densitometric yield determination can be found in Supplementary Fig. 5. b Model for Ub-SUMO2 hybrid chain-mediated recruitment of BRCA1-A complex to double strand breaks (DSBs) through SUMO and Ub modifications of chromatin, presumably H2AX¹². Rap80 contains a SUMO2-interacting motif (SIM) followed by tandem ubiquitin interacting motifs (tUIMs) with high affinity for K63-linked diUb, suggesting the involvement of K63-diUb-SUMO2 hybrid chains. The distinct site of linkage between K63-diUb and SUMO2 is unknown. SUMO2 displays nine possible sites for K63-diUb attachment (the N-terminus and eight lysine residues). c Schematic presentation of the two-step strategy to generate linearly or isopeptide-linked K63-diUb-SUMO2 hybrid chains. Ub-K63GGK bearing the Srt5M motif in its C-terminus (PT/LPT) is reacted with Ub(AT/LAT) in the presence of Srt2A. The resulting K63-diUb is subjected to Srt5M-transpeptidation with a SUMO2 variant bearing either a N-terminal GG sequence or a site-specifically introduced GGK moiety. d Incubation of Srt2A-generated K63-diUb with a SUMO2 variant bearing a diglycine (GG) moiety at its N-terminus in presence of Srt5M leads to formation of the linear K63-diUb-SUMO2 hybrid chain. LC-MS confirms the integrity of the linearly linked hybrid chain. Densitometric analysis revealed linear hybrid chain formation with 78% yield. e SDS-PAGE analysis of Srt5M-mediated transpeptidation of K63-diUb with SUMO2 variants shows specificity for SUMO2-GGK variants. BocK-bearing SUMO2 variants do not yield the respective K63-diUb-SUMO2 hybrid chains. Incubation of K63-diUb and Srt5M in the absence of SUMO2-GGK leads to hydrolysis of the Srt5M recognition motif and hydrolysis of the H₆-tag (depicted by asterisk (*)). UAA stands for unnatural amino acids BocK or GGK. Full gels and densitometrically determined yields as well as LC-MS analysis can be found in Supplementary Fig. 6 and 7a. f Left: Schematic presentation of the two-step strategy to generate K63-diUb-SUMO2 hybrid chains combining enzymatic assembly and sortylation. Enzymatic assembly using Ubc13/Uev1A gives access to K63-diUb bearing the LPT*H motif at the C-terminus of the proximal Ub. Srt5M-mediated transpeptidation with a SUMO2 variant bearing either a N-terminal GG sequence or a site-specifically introduced GGK moiety results in formation of diUb-SUMO2 hybrid chains with wt linkage between the Ub moieties. Right: SDS-PAGE analysis of Srt5M-mediated transpeptidation of wt K63-diUb with SUMO2 variants as well as SDS-PAGE and LC-MS analysis of the purified hybrid chains. Full gels and densitometric yield determination can be found in Supplementary Fig. 7c. Consistent results were obtained over at least three replicate experiments. Source data are provided as a Source Data file.