Fig. 1.

Reporter substrate for analysis of NHEJ. (A) The reporter substrate consists of GFP with an artificially engineered intron, interrupted by an adenoviral exon, flanked by restriction sites for induction of DSBs. In this construct, the GFP gene is inactive; however, upon digestion with HindIII or I-SceI enzymes and successful NHEJ, the construct becomes GFP+. (B) Restriction sites used to introduce DSBs. Digestion with HindIII generates compatible cohesive ends. Because I-SceI has a nonpalindromic 18-bp recognition site, cleavage of the two inverted I-SceI sites generates incompatible ends.