Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair
Figure 1
Total-NHEJ repair between two tandem I-SceI sites results in a variety of products.
(A) EJ5-GFP is shown along with two classes of NHEJ repair products that can restore a GFP expression cassette: one that restores an I-SceI site (I-SceI+), and one that is I-SceI–resistant (I-SceI-). (B) Restoration of the I-SceI site is common in wild-type cells, but undetectable in KU-deficient cells. EJ5-GFP was integrated into HEK293, wild-type ES, and Ku70-/- ES cells. Following transient I-SceI expression in each of these cell lines, GFP+ cells were sorted, and the GFP genes were amplified from these samples using primers depicted in (A). Shown are these products digested with I-SceI or left uncut. (C) The overall frequency of total-NHEJ is unaffected by KU deficiency. Shown is the frequency of repair of EJ5-GFP resulting in GFP+ cells from wild-type and Ku70-/- ES cells transfected in parallel with an I-SceI expression vector. Also shown are Ku70-/- ES cells cotransfected with both I-SceI and KU70 expression vectors.