Fig 6.
DNA-PKcs3A kinase activity is required to repair chromosomal DSBs generated by a zinc finger nuclease. (A) Schematic of the Tcrb locus showing the constant region (Cb) and the enhancer (Eb). The location of the Eb:ZFN cleavage site is indicated, as are the HindIII sites and Eb probe used for Southern blot analysis of Eb:ZFN cleavage. (B) Southern blot analysis of WT, LigIV−/−, and DNA-PKcs3A/3A abl pre-B cells after treatment with imatinib, Eb:ZFN introduction, and treatment with either DMSO (−) or NU7026 (+; i-DNA-PKcs). Genomic DNA was digested with HindIII and probed with the Eb probe. The hybridizing band generated by an Eb:ZFN DSB is indicated, as is the hybridizing band generated by the Tcrb locus, where a DSB was either not generated or was generated and repaired (*). (C) Schematic of the Cel-I assay. NHEJ-mediated repair of Eb-ZFN DSBs can lead to nucleotide loss (boxes). PCR amplification across the Eb-ZFN site followed by denaturation and reannealing will lead to mismatches that can be cleaved by Cel-I. The level of Cel-I cleavage is an underestimate of Eb:ZFN cleavage, as it detects only Eb:ZFN DSBs that have nucleotide deletions upon repair. (D) Cel-I assay of DNA samples from panel B. Full-length and Cel-I-digested products are shown. Note that there were no Cel-I products in the LigIV−/− cells or the DNA-PKcs3A/3A abl pre-B cells treated with NU7026 (+; i-DNA-PKcs), as the Eb:ZFN DSBs generated in these cells were not repaired (see panel B).