Fig. 5: Ubiquitination of TIRR at lysine-187 by DTX3L dictates NHEJ pathway activity and PARP inhibitor sensitivity. | Nature Communications

Fig. 5: Ubiquitination of TIRR at lysine-187 by DTX3L dictates NHEJ pathway activity and PARP inhibitor sensitivity.

From: DTX3L-mediated TIRR nuclear export and degradation regulates DNA repair pathway choice and PARP inhibitor sensitivity

Fig. 5

a Homologous amino acid sequence alignments of NES motif and all the lysine residues (in blue). b In vivo polyubiquitination assay of whole-cell lysates from 293T cells transfected with HA-Ub, Myc-DTX3L, Flag-TIRR-WT, or mutants after MG132 treatment post-IR (8 Gy). WB analysis (c) and quantification (d) of Flag-TIRR protein in whole-cell lysates from 293T cells transfected with Flag-TIRR-WT or K187R mutant after CHX treatment. Data were shown as the mean ± SD from three biological replicates (n = 3). e Co-IP of whole-cell lysates from 293T cells transfected with HA-XPO1, Flag-TIRR-WT, or K187R mutant, harvested after IR (8 Gy). IFC (f) and quantification (g) of cytoplasmic Flag-TIRR-WT or K187R mutant in U2OS cells transfected with indicated constructs after IR (8 Gy). Scale bar, 10 µm. Data were shown as the mean ± SD from 20 fields (>200 cells, n = 20) from three biological replicates. Two-tailed unpaired Student’s t-test; P values based on the order of appearance: 2.41E-26, 0.5355, 0.0533. h–j Control or TIRR knockout U2OS cells were transfected with EV, Flag-TIRR-WT or mutants and treated with IR (8 Gy), followed by WB analysis in whole-cell lysates (h) and IFC of 53BP1(i). 53BP1 foci was quantified (j). Scale bar, 10 µm. Data were shown as the mean ± SD from three biological replicates (n = 200). Two-tailed unpaired Student’s t-test; P values based on the order of appearance: 3.83E-11, 0.1288, 2.75E-62, 0.0988, 0.7984. Analysis of NHEJ (k) and HR (l) activities in control or TIRR knockout U2OS cells transfected with HR or NHEJ reporter constructs in combination with EV, Flag-TIRR-WT, or mutants. Data were shown as the mean ± SD of three independent experiments (n = 3). Two-tailed unpaired Student’s t-test; P values based on the order of appearance: 0.0003, 0.5906, 0.0002, 0.3904, 0.4312 (k); 0.0020, 0.7301, 0.0029, 0.6462, 0.2901 (l). Source data are provided as a Source Data file. Similar results for (b, c, e, h) panels were obtained in three independent experiments.

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