Research Paper Volume 9, Issue 12 pp 2529—2543

POT1 inhibits the efficiency but promotes the fidelity of nonhomologous end joining at non-telomeric DNA regions

Figure 3. POT1 stimulates the recruitment of Artemis to DNA damages sites. (A) POT1 overexpression stimulates the recruitment of Artemis to laser induced DNA damage sites. The recruitment of Artemis is quantified using the software of Leica LAS AF Lite. (B) Mildly knocking down POT1 significantly suppresses the recruitment of Artemis to DNA lesions induced by lasers. The recruitment of Artemis is quantified using the software of Leica LAS AF Lite. (C) POT1 interacts with Artemis upon DNA damages. 293FT cells were co-transfected with a plasmid encoding POT1-FLAG and a vector encoding Artemis-GFP. On day 1 post transfection, cells were irradiated with X-Ray at 6 Gy. At different time points, cells were harvested for immunoprecipitation with an antibody against FLAG, followed by Western blot analysis. (D) Both POT1 and Artemis are recruited to a given DNA damage site. The KillerRed (KR) system is as previously described (25). In brief, it is a fluorescent protein derived from hydrozoan. Long exposure of cells expressing the KR protein generates ROS-induced DNA DSBs. The U2OS reporter cell line harboring ~ 200 copies of TRE elements was co-transfected with myc-POT1 or GFP-Artemis and TA (transcription activator) –KR or TA-cherry. Both TA-KR and TA-cherry proteins may recognize the TRE elements. TA-KR causes DNA damages at the given site while TA-cherry is utilized as a negative control. The transfected cells were then exposed to light, followed by fixation and immunostaining for further analysis. (E) The effect of Artemis overexpression on NHEJ fidelity. The analysis of NHEJ fidelity is as described in Figure 2E.