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 2. POT1 promotes NHEJ fidelity but inhibits NHEJ efficiency. (A) Schematic picture of NHEJ reporter cassette. The reporter and the cell line harboring it are as previously described [7,24]. (B) Expression of FLAG-tagged POT1. (C) The effect of POT1 overexpression on NHEJ efficiency. The NHEJ-I9a was transfected with POT1 vector, I-SceI vector and DsRed for normalizing transfection efficiency using Lonza 4D machine. On day 3 post transfection, cells were harvested for FACS analysis. (D) Overexpressing POT1 sensitizes HCA2-hTERT cells to X-Ray. POT1 overexpressing cells were treated with X-Ray at 4 Gy, and then harvested, reseeded to plates at different numbers. On day 14 post IR, cells were stained with Commassie solution and colonies with at least 50 cells were counted. Cell survival was calculated as the ratio of the relative plating efficiencies of X-Ray treated versus control cells. (E) Analysis of NHEJ fidelity. The method is as previously reported [25]. At least forty clones were used for junction sequencing. bp: base pairs.