Fig 2: Identification of DTX2 substrates.(A) Volcano plot displaying proteins that bind to Myc-DTX2 versus EV. Myc-trap pull-downs were performed in triplicate on whole-cell lysates from HEK293 cells expressing Myc-DTX2 or EV. Proteins shown as blue dots were identified as significantly enriched proteins using a t test with a 5% false discovery rate (FDR) in all three replicates and more abundant in Myc-DTX2 pull-downs compared to EV. Associated with data S1. (B) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 or EV in the presence and absence of ABT-888 using anti-PARP1, anti-SPT16, anti-SSRP1, anti-XRCC5, anti-XRCC6, anti-RPA1, anti–Myc-tag, or anti-actin as indicated. (C) Volcano plot displaying log2 fold change in intensities in peptides modified with Ub from lysates from HEK293 cells expressing GFP-DTX2 compared to EV. Assays were performed in triplicate, and significant changes in ubiquitinated peptide intensities were determined using a Student’s t test with a 1% FDR (permutation-based). Significantly changed ubiquitinated peptides present in all three replicates, more abundant in lysates from cells expressing GFP-DTX2, and from proteins annotated in the Gene Ontology database with the term “DNA repair” are colored as indicated. Associated with data S2. (D) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, EV or Myc-DTX2, and GFP–histone H3.1 (right only) in the presence and absence of ABT-888. The cell lysates and Ni2+–pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. (E) Reduced in vitro autoubiquitination assay showing the formation of GST-2RD-IR-Ubn over time in the presence and absence of ABT-888. IR-Ub, Ub labeled with near-infrared dye. Visualized with the Odyssey CLx Imaging System (LI-COR Biosciences, top) and subsequently by staining with InstantBlue (Expedeon, bottom).

Fig 3: CYREN interaction with Ku in S and G2 inhibits cNHEJ.a, CYREN isoforms. b, Mean percentage of fused ends per metaphase. Error bars, s.e.m. ***P<0.001, *P<0.05, ns non significant. One-way ANOVA, Sidak’s multiple comparison test. n: number of metaphases analysed. Experiment shown is representative of two biological replicates. c, Co-Immunoprecipitation of CYREN-1-3FLAG with Ku70/80 in HEK293T. For gel source data see Supplementary Figure 1. d, Percentage of fusions ± upper and lower value of 95% confidence intervals, Wilson/Brown test. ***P<0.001, **P<0.01, ns non significant. Fisher’s exact test, two-sided. e, Relative abundance of CYREN-1 and 2 mRNA in double Thymidine synchronized cells. f, Immunoblotting of 3FLAG-CYREN-1 in double Thymidine synchronized cells. g. FLAG immunoprecipitation of CYREN1-3FLAG with 2 Gy irradiation followed by FLAG and Ku70 immunoblotting in double Thymidine synchronized cells. *unspecific band. For gel source data see Supplementary Figure 1. Experiment shown is representative of 4 biological replicates. h, Quantification of g. Error bars, s.e.m. over 4 independent experiments. **P<0.01. Ordinary one-way ANOVA.

Fig 4: CYREN interaction with Ku in S and G2 inhibits cNHEJ.a, Immunoblotting of a peptide binding array of full length CYREN-1. Each dot represents 20 aa of CYREN-1 with 19 aa overlap with the previous and following peptide. Upper panel, ponceau. Middle panels, duplicate incubation with Ku70/Ku80 recombinant proteins and immunoblotting with anti-Ku70 antibody. Lower panel, control immunoblotting with anti-Ku70 antibody without incubation with recombinant Ku70/80. For gel source data, see Supplementary Figure 1. b, Alanine scan of CYREN-1 on residues 9 to 46. FLAG immunoprecipitation of protein extracts from HEK293T cells transfected with pCDNA3.1 plasmids expressing wt CYREN-1-FLAG or each of the single residues mutated to Alanine. Total lysate and FLAG-IP were then immunoblotted with anti FLAG or Ku70 antibodies. For gel source data, see Supplementary Figure 1. c, Protein alignment of CYREN-1 Ku Binding Motif among vertebrates. d, Photo-crosslinked pull down of a BPA-BIO-CYREN(2-24) peptide in HEK293T protein extract, followed by immunoblotting with anti-Ku70 and Ku80 antibodies. + and ++, 25µM and 50µM of BPA-Bio-CYREN. 100µM of CYREN(2-24) free peptide was used as a competitor. For gel source data, see Supplementary Figure 1. e, Experimental outline of Fig. 4d. HT1080 6TG cells stably expressing an inducible control GFP or wt or mutant CYREN-1-3xFlag were transduced with shTRF2 on Day 0 and transfected with a control (NT) pool of siRNAs or a pool of siRNAs targeting the 3’UTR of CYREN on Day 2. Expression of CYREN-1 wt and mutants was induced on Day 3 and cells were collected on Day 5. f, FLAG western blot of endogenous C-terminal 3FLAG tagged CYREN-1 and CYREN-2 cells following double thymidine synchronisation. For gel source data, see Supplementary Figure 1.

Fig 5: DNA-PK-dependent detection of cytosolic dsDNA drives STING-independent IRF3-dependent type I IFN responses in cancer cells lacking cGAS T98G cells were treated or not with dsDNA for 6 h prior to immunofluorescence analysis using DNA-PKcs- and pDNA-PKcs-specific antibodies, and DAPI nuclear staining (n = 3 independent experiments). Scale bar, 20 µm.Whole cell extracts from T98G cells were incubated with 80 nt-long biotinylated ssDNA or dsDNA prior to pull-down using streptavidin-affinity beads. Input and eluates were analyzed by WB using indicated antibodies.T98G cells were treated with non-targeting (CTRL) or KU70-targeting siRNAs prior to whole cell extract preparation, and pull-down as in (B). Inputs and eluates were analyzed by WB using indicated antibodies.T98G cells were treated with CTRL or KU70-targeting siRNAs prior to challenge with dsDNA for 6 h. IFNB and CXCL10 mRNA levels were analyzed by RT–qPCR. Graphs present a representative biological triplicate (n = 3 independent experiments).CTRL or STING -/- T98G cells were transfected or not with dsDNA for 8 h prior to analysis of IFNB and CXCL10 mRNA levels (n = 3 independent experiments).As in (E), except that CTRL or IRF3 -/- T98G cells were transfected or not with dsDNA for 6 h prior to analysis (n = 3 independent experiments).CTRL or STING -/- T98G cells were treated or not with CPT for 72 h prior to analyses of IFNB and CXCL10 mRNA levels (n = 3 independent experiments).As in (G), except that CTRL and IRF3 -/- T98G cells were used. Graphs present a representative biological triplicate (n = 3 independent experiments).Schematic representation of cytosolic dsDNA-dependent type I IFN induction in T98G cells. Data information: All immunoblots are representative experiments (n = 3 independent experiments). All graphs present means ± SEM. P-values were determined by Student's t-test. ns: not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.