Colding-Christensen CS , Kakulidis ES , Arroyo-Gomez J , Hendriks IA , Arkinson C , Fábián Z , Gambus A , Mailand N , Duxin JP , Nielsen ML .

NNF13OC0006477 Novo Nordisk Fonden (Novo Nordisk Foundation), R146-A9159-16-S2 Kræftens Bekæmpelse (Danish Cancer Society), EPIC-XS-823839 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020), MR/K007106/1 Medical Research Council

	Fig. 1: UBIMAX efficiently and specifically detects ubiquitin-conjugated proteins in response to DSBs. a Schematic representation of UBIMAX experimental system and workflow. LC-MS/MS; liquid chromatography-tandem mass spectrometry. b His6-ubiquitin was added to extracts at indicated concentrations and analysed by western blot. Arrow indicates concentration of His6-ubiquitin, 0.1 µg/µL, used in subsequent experiments. c, d Extracts were supplemented with DMSO or ubiquitin E1 inhibitor (“E1i”) prior to addition of untagged ubiquitin (“Ub”) or His6-Ubiquitin (“His6-Ub”). Reactions were initiated by addition of buffer (“no DNA”), undamaged plasmid DNA (“DNA”) or linearized plasmid DNA (“DSB”). Samples were analysed by western blot (c) 1 or 30 min after reaction initiation. UBIMAX (experimental outline in d) was performed in independent reaction quadruplicates. Samples were harvested at 30 min and subjected to the UBIMAX workflow outlined in a. no His Ub, untagged ubiquitin; Ub E1i, ubiquitin E1 inhibitor. e Pearson correlation matrix of the experiment outlined in d. Within-replicate mean and ±standard deviation is indicated. no His, untagged ubiquitin. f Mean percent contribution of ubiquitin peptides to summed peptide abundance for UBIMAX samples (“U”), derived from quadruplicate independent reactions, and three biologically independent replicates of a total extract proteome (“TP”). Error bars represent standard deviations. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test for all pairwise comparisons, with those indicated of p-values ˂ 0.0001. no, no DNA; un., undamaged plasmid DNA. g Depth of sequencing illustrated as distribution of log10(iBAQ)-values of ubiquitylated proteins detected by UBIMAX compared to proteins detected in a total extract proteome. Frequency distribution medians (“M”) are shown at the top left corner. iBAQ, intensity-based absolute quantification; a.u., arbitrary units. h Hierarchical clustering analysis of Z-scored ubiquitylated protein abundances robustly changing with DNA treatment. Gene names are provided on the left (or UniProtID if unannotated). Rep., replicate. i Volcano plot analysis comparing ubiquitylated proteins enriched from DSB- versus undamaged DNA-treated samples. Purple 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 FDR ≤ 0.05. Source data are provided as a Source Data file.
	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.
	Fig. 3: DSB-induced ubiquitylation of Dbn1 depends on ATM and is mediated by SCFβ-Trcp1. a Western blot analysis of denaturing His-ubiquitin pulldowns from mock- or Dbn1-immunodepleted extracts supplemented with His6-ubiquitin (“His6-Ub”) prior to addition of linearized plasmid DNA (“DSB”). Immunodepletion control in Supplementary Fig. 3a. PD, pulldown. b, c Western blot analysis of untreated extracts or extracts supplemented with ATM inhibitor (“ATMi”, b) or neddylation E1 inhibitor (“Culi”, c) prior to addition of linearized plasmid DNA. d Western blot analysis of extracts supplemented with the indicated dominant negative Cullin proteins, neddylation E1 inhibitor or buffer prior to addition of linearized plasmid DNA. *unspecific band. e Experimental outline of Dbn1 IP-MS experiment performed in independent reaction quadruplicates. Extracts were untreated or supplemented with ATMi or proteasome inhibitor (MG262) prior to addition of undamaged- (“DNA”) or linearized plasmid DNA (“DSB”). Samples were collected for mock- or Dbn1-immunoprecipitation (IP) at 60 min and analysed by MS. f Volcano plot analysis comparing proteins enriched from DSB-treated Dbn1 IP-MS samples with versus without MG262. Orange and blue dots indicate significantly enriched and -depleted 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. g Scatter plot analysis of the mean abundance difference between proteins enriched with Dbn1-immunoprecipitation from DSB- versus undamaged DNA-treated samples plotted against that of DSB-treated samples without versus with ATMi. Red and blue dots indicate proteins significantly enriched and -depleted with Dbn1-immunoprecipitation in the presence of DSBs. Purple dots/outlines indicate proteins significantly changed in enrichment with Dbn1-immunoprecipitation upon ATMi. Significance was determined by two-tailed Student’s t-test with s0 = 0.1 and permutation-based FDR-control, with 2500 rounds of randomization, to ensure an FDR ≤ 0.05. h Abundance of Skp1, Cul1, Nedd8, and β-Trcp1 across the indicated Dbn1 IP-MS conditions. Horizontal lines indicate the median and significance was determined by one-way ANOVA with Tukeys’s multiple comparisons test for all conditions shown against DSB-treatment. p-values 0.0006, 0.0007, 0.0091, and 0.0062 for DNA versus DSB and p-values 0.0013, 0.009, 0.0241, and 0.007 for DSB+ATMi versus DSB for Skp1, Cul1, Nedd8, and β-Trcp1. a.u., arbitrary units. i Western blot analysis of mock- or β-Trcp1-immunodepleted extracts with addition of β-Trcp1 protein or buffer prior to linearized plasmid DNA. j Cells exposed to 10 Gy ionizing radiation (IR) were lysed after the indicated times, subjected to ubiquitin pulldown and analysed along with whole cell extracts (“input”) by western blot. PD, pulldown; Ub, ubiquitin. k Cells were untreated (“Unt.”) or treated with Culi or ATMi for 1 h before exposure or not to 10 Gy IR, harvested at 30 min, and processed as described in j. l Cells were transfected with control siRNA (siCTRL) or two different β-TRCP1 siRNAs 72 h before exposure or not to 10 Gy IR, harvested at 30 min, and processed as described in j. Source data are provided as a Source Data file.
	Fig. 4: A variant β-Trcp1 degron is necessary and sufficient for inducing Dbn1 and general protein degradation in response to DSBs. a Schematic representation of the conserved variant β-Trcp1 degron in the Dbn1 C-terminus. ADF-H, actin depolymerization factor homology; HCM, helical charged motif; ABD, actin-binding domain. b Extracts were untreated or supplemented with ATM inhibitor (“ATMi”), ubiquitin E1 inhibitor (“Ub E1i”), or neddylation E1 inhibitor (“Culi”), prior to addition of linearized plasmid DNA (“DSB”). Samples were analysed by western blot at the indicated timepoints. c Recombinant Dbn1 WT, S609A, or S609D were added to Dbn1-immunodepleted extracts. Samples were collected from Dbn1-immunodepleted extract prior to addition of recombinant protein, and at the indicated timepoints following addition of protein and linearized plasmid DNA. Samples were analysed by western blot. d Schematic representation of the recombinant proteins generated by insertion of WT or mutated variant β-Trcp1 degron at the M.HpaII C-terminus. e M.HpaII protein with or without the variant β-Trcp1 degron or buffer (“no M.HpaII”) was added to extracts prior to linearized plasmid DNA. Samples were analysed by western blot at the indicated timepoints. f M.HpaII protein with WT or AQ-mutated variant β-Trcp1 degron was added to extracts prior to undamaged- (“DNA”) or linearized plasmid DNA (“DSB”). Samples were analysed by western blot at the indicated timepoints. g In silico analysis of the human proteome revealed numerous proteins containing a potential ATM/ATR-activated β-Trcp1 degron, a subset of which are involved in the DNA damage response. *indicates proteins where the S/TQ site of the putative variant β-Trcp1 degron is known to be phosphorylated. h, i DNA damage, such as DSBs, activates the apical DDR kinase ATM, which mediates phosphorylation of the actin-organizing protein Dbn1 at the S609 SQ motif. This primes the connected conserved variant degron (ii) for recognition by the F-box protein β-Trcp1, resulting in ubiquitylation by the SCFβ-Trcp1 ubiquitin E3 ligase complex and subsequent proteasomal degradation of the Dbn1 protein. Source data are provided as a Source Data file. Figures a, d, g, and h were created with BioRender.com.

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