Fig. 2: Impact of mutations in the BRCT domain of Pol λ on its recruitment to DSBs by Ku.

a Principle of the laser micro-irradiation experiment. U2OS cells engineered for auxin (IAA)-induced Ku70 knockdown, rescued or not with wild-type (WT) Ku70 and expressing either GFP-tagged full-length Pol λ or its BRCT domain, were micro-irradiated with an 800 nm multiphoton laser to generate DSBs in subnuclear areas. b Left panel: representative images before and 20 s after irradiation (irradiated areas are indicated by white arrows) of nuclei from U2OS expressing GFP-tagged full-length Pol λ and depleted or not of Ku70 (±Ku). Scale bars represent 10 µm. Right panel: quantification of fluorescence accumulation at laser-induced DNA damage sites. Results are plotted as mean values ± SEM of n = 21 and n = 34 irradiated nuclei in the presence or absence of Ku, respectively. c Same as in (b) with U2OS expressing GFP-tagged Pol λ-BRCT. Results are plotted as mean values ± SEM of n = 27 and n = 22 irradiated nuclei in the presence or absence of Ku, respectively. d Position of mutated residues in the BRCT domain of Pol λ (purple) at the interface with Ku70/Ku80 (orange and green, respectively). e Quantification of fluorescence accumulation at laser-induced DNA damage sites in U2OS cells expressing GFP-tagged WT or mutated BRCT domain of Pol λ. Results are plotted as mean values ± SEM of 20 to 47 irradiated nuclei (Polλ-BRCT-WT: n = 47; -R57A: n = 20; -R57E: n = 20; -L60A: n = 23; -L60R: n = 21; -F61G: n = 20; -R96A: n = 22; -S116G: n = 22). Source data are provided as a Source Data file.