Fig. 2. UBIMAX identifies DNA damage specific ubiquitylation events and detects DSB-induced ubiquitylation of Dbn1.

a Experimental outline for the UBIMAX experiment profiling ubiquitylated proteins in response to DPC-containing substrates. Extracts were untreated or supplemented with ubiquitin E1 inhibitor (“Ub E1i”) prior to addition of His₆-Ubiquitin (“His₆-Ub”). Reactions were initiated by addition of buffer (“no DNA”), undamaged plasmid DNA (“DNA”), plasmids carrying the M.HpaII protein crosslinked at a single-stranded DNA gap (“ssDNA-DPC”), or plasmids carrying the Flp protein crosslinked at a single-strand break (“SSB-DPC”). Reactions were performed in independent reaction triplicates. Samples were harvested at 30 min and subjected to the UBIMAX workflow outlined in Fig. 1a. b, c Volcano plot analysis comparing ubiquitylated proteins enriched from ssDNA-DPC (b) or SSB-DPC (c) versus DNA-treated samples. Pink/orange and blue dots indicate significantly enriched and -depleted ubiquitylated proteins. Significance was determined by two-tailed Student’s t-test, with permutation-based FDR-control with s0 = 0.1 and 2500 rounds of randomization, to ensure an FDR ≤ 0.05. Ubiquitylated proteins with FDR ≤ 0.01 are labelled. d–i Abundance of Ku80 (d) Ku70 (e) Mre11 (f) Rpa1 (g) Chfr (h) and Dbn1 (i) across ubiquitin target enriched samples of the UBIMAX experiments profiling protein ubiquitylation in response to DSBs (Fig. 1d) and DPCs (a). Horizontal lines indicate the median and significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test for all conditions against undamaged DNA with a cut-off of p-value ≤ 0.01 and with those indicated of p-values ˂ 0.0001. n = 3 independent reaction replicates for DSB and DPC conditions and n = 7 for no DNA and DNA conditions. a.u., arbitrary units. j Extracts were untreated or supplemented with ubiquitin E1 inhibitor prior to initiation of reactions by addition of undamaged plasmid DNA (“DNA”), linearized plasmid DNA (“DSB”), or plasmids carrying a DPC at a ssDNA gap (“ssDNA-DPC”). Samples were analysed by western blot at the indicated times. *unspecific band. Source data are provided as a Source Data file.