Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair
Figure 2
(A) EJ2-GFP is shown with 3 NHEJ products that are found to result in GFP+ cells. Shown in the ovals are the relative contributions of these products, based on the analysis shown in (B). The predominant GFP+ product, labeled Xcm1+, uses 8 nts of homology flanking the I-SceI site to generate an XCM1 site, resulting in a 35 nt deletion. (B) Analysis of EJ2-GFP repair products that restore the GFP+ gene. EJ2-GFP was integrated into HEK293, wild-type ES, and Ku70-/- ES cells. Using the primers shown in (A), the GFP genes were amplified from the parental ES EJ2-GFP cell line, and also from sorted GFP+ cells from each of the above cell lines following transient I-SceI expression. Shown are these amplification products, which were either uncut or cut with XCM1. (C) Repair by alt-NHEJ (EJ2-GFP) is suppressed by KU. Shown are the frequencies of alt-NHEJ repair, following transient I-SceI expression, for the wild-type and Ku70-/- EJ2-GFP cell lines, along with the Ku70-/- line co-transfected with an expression vector for KU70. Also shown are parallel experiments with the SA-GFP and DR-GFP reporters (see Figure 3). Asterisks denote a statistical difference in repair efficiency between Ku70-/- versus both wild-type, as well as Ku70-/- with transient expression of KU70 (p<0.0005).