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 1. POT1 is rapidly recruited to nontelomeric DNA damage sites. (A) POT1 is recruited to DNA damage sites within 1-second post microirradiation. U2OS cells were microirradiated to generate DSBs in a line pattern using a 405 nm diode laser. (B) Diagram of the site for which ChIP primers were designed (arrows). At different time points post I-SceI transfection, NHEJ-I9a cells were harvested and lysed for ChIP assay with an antibody against POT1, followed by quantitative PCR analysis. The procedure for ChIP is as previously described [12]. (C) Schematic depiction of different domains of POT1 tagged with GFP. (D) Comparable expression of GFP-tagged different domains of POT1. The U2OS cells were transfected with different amounts of vectors encoding OB1 (0.67 μg), OB2 (0.5 μg), OB12 (1 μg), C-terminal (3 μg). At 48 h post transfection, cells were harvested for FACS analysis. (E) Analysis of recruitment of different POT1 domains.