An E3 ubiquitin ligase localization screen uncovers DTX2 as a novel ADP-ribosylation-dependent regulator of DNA double-strand break repair

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair. DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair. Maintenance of genome stability is central to cell homeostasis, organismal development, and reproduction (1Panier S. Wang S. Schumacher B. Genome Instability and DNA repair in somatic and reproductive aging.Annu. Rev. Pathol. Mech. Dis. 2024; 19: 261-290Crossref PubMed Scopus (3) Google Scholar). Therefore, all living organisms rely on complex signaling pathways to prevent, detect, signal, and repair the myriad of DNA lesions that threaten the integrity of their genetic material on a daily basis (2Ciccia A. Elledge S.J. The DNA damage response: making it safe to play with knives.Mol. Cell. 2010; 40: 179-204Abstract Full Text Full Text PDF PubMed Scopus (3307) Google Scholar, 3Thada V. Greenberg R.A. Unpaved roads: how the DNA damage response navigates endogenous genotoxins.DNA Repair (Amst). 2022; 118103383Crossref PubMed Scopus (5) Google Scholar). 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We find that DTX2 recruitment to DNA breaks is primarily mediated by its tandem WWE domains with additional contribution from its characteristic C-terminal DELTEX and RING domains. DTX2 depletion impedes BRCA1 foci formation and decreases the efficiency of HR repair. In agreement with these findings, DTX2-depleted cells are sensitive to X-ray irradiation and PARP inhibition, formally demonstrating the functional relevance of DTX2 in DNA repair and cell tolerance to genotoxic stress. To uncover novel E3 ubiquitin ligases involved in the DDR, we surveyed online databases to define the nuclear RING/U-box E3 ubiquitin ligase landscape of human cells (Fig. 1A and Table S1). We first extracted all proteins annotated as nuclear from four databases: (1) The Human Protein Atlas (58Thul P.J. Akesson L. Wiking M. Mahdessian D. Geladaki A. Ait Blal H. et al.A subcellular map of the human proteome.Science. 2017; 356eaal3321Crossref PubMed Scopus (1769) Google Scholar), (2) UniProt (59Bateman A. 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Chem. 2014; 289: 6619-6626Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Phosphorylated H2A.X (γ-H2A.X) and the RPA 32 kDa subunit (RPA32) were used for colabeling as DNA damage markers for early (5 min) and late (60 and 180 min) microirradiation time points, respectively (Fig. 1, D and E). Out of all 57 tested candidates, 50 were well-expressed but did not form detectable stripes. We also identified seven novel E3 ligases that formed clear stripes in microirradiated nuclei: DTX2, DTX3, PCGF6, PHF21A, ZNF48, RNF34, and RNF114 (Figs. 1F, S1, A–C and Table S3). Here, we chose to concentrate our characterization efforts on the DELTEX family protein, DTX2, as its recruitment was the most profound and striking among all candidates (Figs. 2, A and B and S1, A–C).Figure 2DTX2 localizes to microirradiation stripes in a PARP-dependent manner. A, schematic representation of the 2 DTX2 isoforms and immunoblot of U-2 OS total cell extracts. The protein region corresponding to exon 4 is presented in red and is absent from DTX2B. B and C, DTX2A/B are recruited to microirradiation stripes. U-2 OS cells were individually transduced with lentiviruses encoding HA-tagged DTX2A/B. Subsequently, 48 h post selection, cells were microirradiated, and IF staining was achieved for HA and γ-H2A.X as early (5 min) or RPA32 as late (60/180 min) DNA damage markers. Quantification of microirradiated cells with DTX2A/B accumulation at positive tracks. Data represent the mean % of cells with DTX2A/B/γ-H2A.X or RPA32-colocalizing stripes ± SD (n = 3 biological replicates). D and E, kinetics of endogenous DTX2 recruitment at different time points (min) to microirradiation stripes ± SD (n = 3 biological replicates) (antibody validation in Fig. S2, A–C). F and G, PARP inhibition abrogates endogenous DTX2 recruitment to DNA damage. Cells were treated with vehicle (DMSO) or 5 μM PARPi (AZD-2281, olaparib) for 30 min prior to performing microirradiation and IF as in (B). Data represent the mean % of cells with endo DTX2/γ-H2A.X colocalizing stripes ± SD (n = 4 biological replicates). Statistical significance was established using an unpaired t test (p < 0.0001 (∗∗∗∗)). H and I influence of PARylation on DTX2 recruitment to microirradiation stripes. U-2 OS cells were transfected with siRNAs targeting PARG, PARP1, PARP2 or both and 48 h later, microirradiation and IF was performed. Data represent the mean % of cells with endo DTX2/γ-H2A.X colocalizing stripes ± SD (n = 3 biological replicates). In the bar graphs, each data point represents an independent biological replicate. The scale bar represents 10 μm. IF, immunofluorescence; PARG, poly(ADP-ribose) glycohydrolase; DMSO, dimeth