Figure 5. The absence of Ku86 results in predominately microhomology-based end joining. (A) HindIII-linearized pEGFP- Pem1-Ad2 plasmids were recovered from either WT HCT116, Ku86+/2 or Ku862/2 cells, propagated through E. coli and then analyzed for retention of a single HindIII restriction site (‘‘perfect rejoining’’) by HindIII restriction enzyme digestion analysis. The asterisks indicate those plasmids where perfect rejoining occurred. (B) The results of four or more experiments similar to those depicted in (A) were combined and summarized. N.B. The re-expression of a WT DNA-PKcs or XLF cDNA (+cDNA) in their respective cell lines reduced the frequency of perfect rejoining back to WT levels, confirming the specificity of the effect. doi:10.1371/journal.pgen.1000855.g005 above results with Ku86-null cells, we next tested whether the residual repair in these other C-NHEJ-null cell lines was also heavily biased towards microhomology. Indeed, although there were far fewer repair events in these three cell lines in comparison to Ku86-null cells, they were nonetheless predominantly (70% to 80%) mediated by microhomology (Figure 5B). Thus, DNA-PKcs-, XLF- and LIGIV-null cell lines had an identical repair profile to Ku86-null cells (Figure 5B), but carried out only 1% to 10% as many repair events as Ku86-null cells. A-NHEJ is negatively regulated by Ku in human somatic cells The above results suggested that Ku normally actively suppresses A-NHEJ. In Ku’s presence, even when C-NHEJ is inactivated by mutations in DNA-PKcs, XLF or LIGIV, A-NHEJ is apparently still strongly suppressed (Figure 3). In contrast, in Ku’s absence, A-NHEJ is ‘‘unleashed’’ and rescues the repair activity of the cells (Figure 4). Interestingly, there is precedent for this model in the literature. Thus, mouse cell lines deficient for Ku86 repaired I-SceI-induced DSBs with a frequency similar to Ku86 Regulates Pathway Choice in Human Cells that of wild-type cells but with a repair profile that was biased towards microhomology [17–21,64]. Moreover, the ionizing radiation sensitivity of LIGIV-deficient chicken DT40 cells can be rescued by the deletion of Ku70 [65]. Perhaps most impressively, LIGIV deficiency in the mouse results in embryonic lethality and this can be rescued by the deletion of Ku86 [66]. Although, repair profiles were not assessed in the latter two studies, they are consistent with the absence of Ku de-repressing A-NHEJ to the point where the phenotypes could be rescued. To investigate if this paradigm could be extended to human cells we directly tested whether the strong repair defects of DNA- PKcs- and the very severe defects of LIGIV-null cells could be rescued by reducing the amount of Ku in these cell lines. A combination of genetic and molecular approaches was utilized to achieve a significant knockdown of Ku, a highly abundant protein. Thus, rAAV gene targeting was first used to functionally inactivate one Ku70 allele in DNA-PKcs- and LigIV-null cell lines. DNA- PKcs2/2:Ku70+/2 and LIGIV2/2:Ku70+/2 cell lines have ,50% the level of Ku70 protein compared to wild-type cells (Figure 6B; [42,50]) and this reduction in Ku slightly rescued the repair deficiencies of either cell line (compare panel 5 with panel 6 and panel 8 with panel 9 in Figure 6A; Figure 6C). siRNA against Ku70 was then used to reduce the level of Ku protein to ,5% of wild-type (+siRNA, Figure 6B). Impressively, DNA-PKcs2/2 and LIGIV2/2 cells showed wild-type and greatly enhanced, respec- tively, end-joining activity (compare panel 5 with panel 7 and panel 8 with panel 10 in Figure 6A; Figure 6C), directly demonstrating that a reduction in Ku can ‘‘reanimate’’ a cell that appears ‘‘dead’’ for DNA DSB repair. Importantly, the end joining occurring in these Ku-reduced cell lines was predominately microhomology mediated (Tables S3, S4, S7, S8). Moreover, these data provide a plausible molecular mechanistic explanation for the earlier genetic results obtained in chickens and mice. Microhomology-mediated A-NHEJ predominates in C-NHEJ deficient cells To confirm and extend the above results, we utilized a reporter assay that is biased towards detecting A-NHEJ events. pDVG94 is designed such that the relative efficiency of C-NHEJ versus A- NHEJ events can be assessed [26,67]. When pDVG94 is digested with AfeI and EcoRV it results in a blunt-ended linear substrate with a 6-bp repeat at both ends (Figure 7A). C-NHEJ can rejoin these ends and yield a wide variety of junctions but A-NHEJ almost exclusively generates a single product in which the 2 repeats have been reduced to 1, which simultaneously generates a novel BstXI restriction enzyme recognition site (Figure 7A). Thus, linearized pDVG94 plasmid was transfected into the mutant cell lines and 48 hr later repaired plasmids were recovered, purified and then used as substrates for PCR using a 59-radiolabeled PCR primer (Figure 7B). The relative level of A-NHEJ is subsequently determined by quantification of the BstXI-digested PCR products where a 180 bp product represents the repaired plasmid and a cleaved 120 bp product is diagnostic of microhomology-mediated end joining (Figure 7B). The parental cell line, Ku86flox/2 and Ku86flox/2 infected with AdCMV cell lines carried out only a few percent of microhomology-mediated end joining in this assay (Figure 7C). In contrast, Ku86-null cells showed on average 45% microhomology use (Figure 7C). Although this assay cannot be used to determine the absolute frequency of the individual repair events, it confirmed that in the absence of Ku, microhomology- mediated events became easily detectable. This phenotype was even more evident in the DNA-PKcs- (Figure 7D), XLF- (Figure 7E) and LIGIV- (Figure 7F) cell lines. In the absence of one of these three factors, the frequency of PLoS Genetics | www.plosgenetics.org 6 February 2010 | Volume 6 | Issue 2 | e1000855