FIG. 4.

Inefficient activation of DNA-PK by nicked DNA. (A) Nicked hairpin-ended DNA. DNA-PK was tested for activation by 68-bp nicked hairpin-ended DNA or 70-bp open-ended DNA. (B) Nicked plasmid DNA. DNA-PK (70% pure) was tested for activation by untreated supercoiled plasmid DNA (closed circles), plasmid linearized with EcoRI (closed squares), plasmid partially nicked with DNase I (open triangles), plasmid nicked with DNase I and linearized with EcoRI (open squares), an equal mixture of untreated plasmid and plasmid linearized with EcoRI (open circles), or an equal mixture of plasmid nicked with DNase I and plasmid linearized with EcoRI (closed triangles). For the DNA mixtures, each type of DNA was added in the amount indicated on the graph. (C) Analysis of nicked plasmid DNA. DNA preparations were analyzed by electrophoresis in a 1% agarose gel containing ethidium bromide, which resolved nicked, linear, and negatively and positively supercoiled (sc) plasmid DNA at the indicated positions. Negatively supercoiled plasmid DNA was left untreated (lane 1), linearized with EcoRI (lane 2), or partially nicked with DNase I (lane 3). The DNase I-treated plasmid was converted to nicked linear DNA by EcoRI (lane 4), not affected by incubation with DNA-PK (lane 5), and contained very few lesions other than simple nicks, since T4 DNA ligase converted nearly all of the nicked DNA to covalently closed circular DNA that migrated as positively supercoiled DNA (lane 6). Similar results were obtained with 95% pure DNA-PK.