Figure 3. The loss of C-NHEJ greatly reduces end joining. The indicated cell lines were transfected with HindIII- (Top panels) or I-SceI- (Bottom panels) linearized pEGFP-Pem1-Ad2 plasmid together with a pCherry plasmid. The cells were analyzed by FACS 24 hr post transfection. The number of cells that were doubly EGFP (horizontal) and pCherry (vertical) positive versus the number that were pCherry positive was determined. For a given experiment the data are shown as percent repair in the upper right-hand corner of each plot. doi:10.1371/journal.pgen.1000855.g003 an end-joining activity that was 90% of wild-type (Figure 2) directly demonstrating that the absence of XLF was responsible for the phenotype of the cells. In conclusion, these experiments demonstrated that C-NHEJ is the major NHEJ repair pathway in human somatic cells and in its absence only low (albeit detectable—see below) levels of end joining can occur. The absence of Ku changes the repair profile, but not the repair activity Primates are unique in that, in contrast to every other species examined to date, the Ku genes have evolved to become essential [40,42,44], due to their ability to suppress lethal HR-mediated telomere recombination [43]. Consequently, human cell lines that are for either Ku70 [42] or Ku86 [40] are not viable. We have, however, constructed a ‘‘conditionally null’’ (Ku86flox/2) cell line for Ku86. This cell line has been engineered through rAAV gene targeting technology to contain only a singly functional ‘‘floxed’’ allele of Ku86 [44]. In the presence of the Cre recombinase, the floxed allele is excised and the cells become for Ku expression. Importantly, the loss of Ku86 is essentially complete in 3 to 4 days and although the cells will ultimately succumb, they generally don’t do so for approximately 2 weeks [44]. Thus, the day 4 to day 14 window was used to assess the ability of the cells to perform end joining. Consequently, Ku86flox/2 cells were either infected with a control adenovirus (AdCMV) or an adenovirus expressing Cre (AdCre). At 4, 5 and 6 days post- infection, a portion of the cells were processed for Western analysis, which confirmed that the levels of Ku86 protein were greatly diminished in the AdCre-treated cells compared to the control AdCMV-treated cells (Figure 4A). The levels of Ku86 never go to zero because a minor portion of the cells are either not productively infected with the adenoviral vector and/or they do not efficiently undergo Cre-mediated recombination [44]. At 120 hr post adenovirus infection, the cells were transfected with pEGFP- Pem1-Ad2 and 24 hr later the cells were analyzed by FACS analysis. Very unexpectedly, Ku86flox/2 +AdCre cells performed Ku86 Regulates Pathway Choice in Human Cells Figure 4. Ku86-null cells show wild-type levels of end joining activity. (A) Western blot analysis shows that the expression of Cre (AdCre) in Ku86flox/2 cells results in the reduction of Ku86 expression. AdCMV is a negative control adenoviral vector. (B) the indicated cell lines were transfected with either HindIII- (Top panels) or I-SceI- (bottom panels) linearized pEGFP-Pem1-Ad2 plasmid. All symbols are as in Figure 3. doi:10.1371/journal.pgen.1000855.g004 end joining at a wild-type frequency (Figure 4B). Indeed, in four independent experiments the ‘‘Ku86-null’’ cells reproducibly seemed to have even slightly higher levels of end joining activity than wild-type cells, regardless of whether HindIII- or I-SceI- linearized substrates were used (Figure 2). We were perplexed by this result until we considered the possibility that although the frequency of end-joining was not altered in Ku86-null cells the repair profile might be. To experimentally test this hypothesis, the repaired HindIII-cleaved pEGFP-Pem1-Ad2 substrate plasmids were recovered from Ku86- cells and analyzed by agarose gel electrophoresis following HindIII re-digestion for perfect rejoining. In the parental and heterozygous cell lines this type of repair event was observed in about 30% of the repaired plasmids (asterisked lanes in Figure 5A and 5B). In striking contrast, ,80% of all the plasmids recovered from Ku86-null cells had reconstituted a single HindIII site (Figure 5A and 5B). Thus, while the overall repair frequency in Ku86-null cells was not significantly different from wild-type cells, the repair profile was heavily shifted to one that utilized more microhomology. Microhomology-mediated end joining also dominates in the absence of DNA-PKcs, XLF, and LIGIV Although end joining was greatly reduced in DNA-PKcs-, XLF- and LIGIV-null cell lines (Figure 3), it was not zero. Given the PLoS Genetics | www.plosgenetics.org 5 February 2010 | Volume 6 | Issue 2 | e1000855