Fig. 6: CXorf56 protein binds to Ku70 DNA binding domain in S and G2 phases, impeding the recruitment of Ku70 and its downstream responders to DNA damage sites.

A LMNA (lamin A) Cas9 reporter. CRISPR-Cas9 and sgRNA target the 5’ UTR of LMNA. Cas9 with sgRNA generates DSBs. HR fuses mClover with a start codon (red arrow) to LMNA for repair. mClover protein levels (B) were investigated by western blotting and the average percentage of mClover+ cells (C) in indicated MDA-MB-231 cells in five independent experiments, normalized to the vector control (Vec ctrl) group. Data were analyzed by a two-tailed t test. D, E Corresponding MDA-MB-231 cells were treated with IR (1 Gy, 1 hour for DNA-PKcs, XRCC4, and LIG4), and indicated foci were detected by immunofluorescence. Representative images are shown in D, Scale bars = 10 μm., and quantification of focus signals is shown in E. Data were analyzed by a two-tailed t test. F–H MDA-MB-231 cells were synchronized with nocodazole (100 ng/ml) for 12 hours and released into the cell cycle. At the indicated time points, cells were harvested for cell cycle (F) and co-immunoprecipitation and Western blotting analysis without IR treatment (G) or with 1-hour of IR treatment (2 Gy) (H). I Schematic of the domains of Ku70. There are three main domains in Ku70: the N-terminal vWA domain (blue), the core DNA binding domain (DBD, yellow), and the C-terminal domain (CTD, gray). J MDA-MB-231 cells were transfected with plasmids encoding the indicated Flag-tagged truncated Ku70 constructs. Cell lysates were subjected to immunoprecipitation with an anti-Cxorf56 protein antibody. Precipitated proteins were analyzed by Western blotting with an anti-Flag antibody. Input controls were also included. Remarks: ^nsp ≥ 0.05, *p < 0.05, **p < 0.01; Data are presented as mean ± SD.