Fig. 4: Analysis of Rad52- and SUMO-dependent interactome.

a A schematic drawing depicting identification of potentially SUMOylated proteins at the sites of Rad52 repair by Split-TurboID (upper panel). In this strategy, the N-terminal, small Split-TurboID fragment is fused to SUMO or SUMO-KallR, and the C-terminal, large fragment to a protein of interest (here Rad52). Target SUMOylation (or SUMO-SIM interaction) results in protein-fragment complementation and reconstitution of the TurboID enzyme, allowing specific biotin proximity labeling of interactors, and other proximal proteins, in a SUMO-dependent manner. Biotinylated proteins (X, Y, Z) can then be enriched by streptavidin purification and identified by mass spectrometry (MS). In the lower panel, schematic representation of the genetic structure of Split-TurboID tagged constructs is also shown. b Streptavidin pulldown of biotinylated proteins in indicated transformants. Western blot analysis of total cell extracts (INPUT, left side) and streptavidin pulldown (IP, right side) prepared from a control untagged (WT) and the Rad52-12Pk-CTurbo (Rad52-C) strains transformed with an empty vector or 6HA-N-Turbo-NSUMO (N-SUMO) bearing plasmid was performed using anti-biotin (upper panel), and then the membrane was stripped, cut and developed with anti-V5 (middle panel), and anti-HA (lower panel) antibody. A Ponceau S-stained blots were added to show equal amounts of total proteins loaded onto the gel after isolation from individual transformant. Representative blots from n = 3 biological replicates. All INPUTs and IPs blots are cut and merged from shorter and longer exposures of the same membrane. c, d Results of three independent biological experiments followed by proteomics analyses of the proximal biotinylation assay obtained for NTurbo-SUMO-ID (c) or NTurbo-SUMO-KallR-ID (d) in the WT and SUMO-KallR background, respectively. Proteins significantly enriched in comparison to controls (top right-hand side) are identified as possible Rad52 interactors and potential SUMOylation targets at endogenous replication stress sites. All hits were annotated according to their cellular function, process, and location based on GO Terms annotations database. Annotations were then slimmed into broader categories, and the categories of interest are shown on the plot. Statistical significance was calculated using two-sided Student’s t-test without an adjustment for multiple comparisons, but with an effect size cut-off applied. Source data are provided as a Source data file.