Reporter constructs for analysis of DSB repair. (a) Reporter cassette for detection of NHEJ of compatible DNA ends (NHEJ-C). The cassette consists of a GFP gene under a CMV promoter with an engineered intron from the rat Pem1 gene, interrupted by an adenoviral exon (Ad). The adenoviral exon is flanked by I-SceI recognition sites in direct orientation for induction of DSBs. In this construct the GFP gene is inactive; however upon induction of a DSB and successful NHEJ the construct becomes GFP+. SD, splice donor; SA, splice acceptor; shaded squares, polyadenylation sites. (b) Reporter cassette for detection of NHEJ of incompatible DNA ends (NHEJ-I). The cassette is similar to the one shown above, but the I-SceI sites are in inverted orientation, therefore I-SceI digestion produces incompatible ends. (c) Reporter cassette for detection of HR. The cassette consists of two mutated copies of GFP-Pem1. In the first copy of GFP-Pem1 the first GFP exon contains a deletion of 22 nt and an insertion of two I-SceI recognition sites in inverted orientation. The 22 nt deletion ensures that GFP cannot be reconstituted by a NHEJ event. The second copy of GFP-Pem1 lacks the ATG and the second exon of GFP. Upon induction of DSBs by I-SceI, gene conversion events reconstitute an active GFP gene. (d) Compatible DNA ends generated by digestion of two I-SceI sites in direct orientation. (e) Incompatible DNA ends generated by digestion of two inverted I-SceI sites.

Comparison of the efficiencies of NHEJ and HR across different chromosomal locations. For each DSB repair pathway 7 HCA2-hTERT cell lines, each containing a single randomly integrated copy of the NHEJ-C, NHEJ-I, or HR reporter cassettes were analyzed. Cell lines were co-transfected with the plasmids I-SceI (5µg), and pDsRed2-N1 (0.1µg). I-SceI endonuclease generates DSBs in the GFP-based reporter cassettes (Fig. 1) and successful repair by NHEJ, or HR by gene conversion results in the appearance of GFP+ cells. To quantify NHEJ or HR events the cells were analyzed by flow cytometry 4 days after transfection. Transfection efficiency was normalized by DsRed. The ratio of GFP+ to DsRed+ cells was used as a measure of NHEJ or HR. Typically 20,000 cells were analyzed for each sample; in the treatments where the numbers of GFP+ cells were low, 40,000 cells were scored. The experiments were repeated at least five times and error bars are S.D.

NHEJ is a faster process than HR. (a) Western blot showing kinetics of expression of I-SceI after Amaxa transfection. Time indicates hours after transfection. (b) Kinetics of GFP expression following Amaxa transfection. Cells were harvested at indicated time points after transfection and analyzed by flow cytometry. (c) Kinetics of NHEJ and HR. Two representative reporter cell lines containing each type of construct were chosen for this analysis: NHEJ-C, lines S30c and S44c; NHEJ-I, lines I-26c and I-29c; HR, lines H9b and H15c. Cells were transfected with the plasmid encoding I-SceI endonuclease to induce DSBs. For each cell line multiple transfections were pooled, cells were mixed and plated on multiple plates to eliminate variation in transfection efficiency between time points. Cells were harvested at indicated time points post-transfection and analyzed by flow cytometry. GFP+ cells correspond to DSB repair events. The absolute values for DSB repair differ from those reported for the corresponding clones in Fig. 2, because the data in this experiment is not corrected for transfection efficiency with DsRed. Typically, 20,000 cells were analyzed in each sample; in the treatments where the numbers of GFP+ cells were low, 40,000 cells were scored. The experiments were repeated at least four times and error bars are S.D.