Ku86 Regulates Pathway Choice in Human Cells Figure 1. Reporter substrate for analysis of NHEJ. A cartoon of the reporter construct (pEGFP-Pem1-Ad2). (A) The construct is essentially a GFP cassette whose expression is driven by CMV promoter and terminated by the SV40 polyA sequence. ‘‘G’’ is separated from ‘‘FP’’ by a 2.4 kb intron containing an exon (Ad) from adenovirus that is flanked by HindIII and I-SceI restriction sites. Splice donor (SD) and splice acceptor (SA) sites are shown. (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. (C) Due to the presence of the Ad-exon into the middle of the Pem1 intron, the Ad exon is efficiently spliced into the middle of the GFP ORF, inactivating the GFP activity and thus making the starting substrate GFP negative. Both sides of the Ad exon have HindIII/I-SceI restriction sites. Cleavage with either of these endonucleases removes the Ad exon and upon successful intracellular plasmid circularization GFP expression is restored and can be quantitated by flow cytometry. doi:10.1371/journal.pgen.1000855.g001 engineered such that it is interrupted by a 2.4 kb intron derived from the rat Pem1 gene (Figure 1A). An exon derived from adenovirus (Ad2) has been introduced into the middle of the intron and it is flanked on both sides by HindIII and I-SceI restriction enzyme recognition sequences. In the unmodified configuration, GFP is not expressed because the Ad2 exon is efficiently incorporated into the GFP mRNA (Figure 1C). Digestion of the plasmid either with HindIII or I-SceI at the flanking sites generates a linear plasmid lacking the adenoviral exon with either compatible 59-overhanging cohesive ends or incompatible ends, respectively (Figure 1B). The HindIII sites are arranged such that cohesive 4-bp overlapping ends are generated, whereas the I-SceI sites are arranged in an inverted orientation, which demands that some sort of processing must occur before the ends can be rejoined. Thus, the impact of loss-of-function NHEJ gene mutations on these aspects of end joining can be individually assessed. Un-digested or partially digested plasmids, because of the retention of the Ad2 exon, generate a product unable to express GFP. Due to the buffering capacity of the intron, end joining by the cellular repair apparatus of transfected, linearized plasmid usually re-constitutes GFP expression, even when extensive additions or deletions of nucleotides have occurred (Figure 1C). As a result, a wide spectrum of end joining events can be detected and quantitated by FACS (fluorescently activated cell sorting). As a transfection control, cells are always co-transfected with a pCherry expression plasmid and the data are expressed as the percentage of cherry-positive cells that are also green-positive. Lastly, pEGFP- Pem1-Ad2 contains a bacterial origin of replication and an antibiotic resistance gene permitting the plasmids to be recovered from human cells and rescued in E. coli. Consequently, the structure of the repair junctions, which provides mechanistic insight into the type of repair that was utilized, can be identified by DNA sequencing. This assay system was used to interrogate a series of isogenic human HCT116 cell lines. The derivative cell lines were engineered using rAAV gene targeting [46,49] to be reduced or deficient in the expression of most of the C-NHEJ factors, namely: Ku70 [42,50], Ku86 [40,44], DNA-PKcs [51], XLF (Fattah et al., unpublished) or LIGIV (Oh et al., unpublished). HCT116 is a human adenocarcinoma somatic tissue culture cell line that is mismatch repair defective [52,53] and it is important to note that defects in mismatch repair genes have been implicated in affecting both HR- and NHEJ-mediated DSB repair efficacy {[54,55] reviewed by [56]}. Moreover, it is also relevant that the cell line contains reduced levels of Mre11, Rad50 and Nbs1 [57,58], a trimeric complex of proteins that have documented roles in HR and which have recently also been implicated in both C- and A- NHEJ {[59–61]; reviewed in [38]}. Despite these deficiencies, HCT116 is only slightly reduced for general NHEJ activity [62]. In addition, it is diploid, has a stable karyotype, exhibits normal DNA damage checkpoints and is wild-type for most of the other major DNA DSB repair genes [47]. These facts, combined with the previous extensive use of HCT116 in gene targeting studies [47] recommended this cell line for these analyses. LIGIV is the only C-NHEJ gene that is haploinsufficient for plasmid DNA end joining in vivo When HindIII- or I-SceI-linearized pEGFP-Pem1-Ad2 plasmid was introduced into the parental HCT116 cell line, intracellular circularization allowing GFP expression could easily be detected and quantitated by flow cytometry (Figure S1). When the same experiment was carried out with the C-NHEJ heterozygous cell lines, significant repair activity was always observed (Figure S1). Averaged over four experiments, the Ku70, Ku86, DNA-PKcs and XLF heterozygous cell lines showed only a slightly reduced ability to repair this DSB that was not significantly different from wild- PLoS Genetics | www.plosgenetics.org 3 February 2010 | Volume 6 | Issue 2 | e1000855