These experiments demonstrated that in the presence of Ku, but in the absence of other C-NHEJ factors, that virtually all of the end joining in human cells is carried out by microhomology- mediated processes. In contrast, in the presence of the other C- NHEJ factors, but in the absence of Ku, some, but not all (see the Discussion), of the end joining occurs using microhomology. Ku protects DNA ends from degradation In every metabolic reaction (e.g., DSB repair, V(D)J recombi- nation, telomere maintenance, etc.) that Ku participates in, and in every organism that such reactions have been characterized, Ku’s absence is marked by hyper-resection of the relevant DNA ends [11]. To determine if this aspect of Ku’s absence is conserved in human cells extensive sequencing was carried out of pEGFP- Pem1-Ad2 plasmids recovered from wild type and Ku86-null cells. A significant increase in deletion size in Ku86-null cells compared to wild-type cells was observed. In wild-type cells the median deletion size was 595 bp whereas in Ku86-null cells it was 1158 bp for HindIII-linearized plasmids (Table S1 and Figure S4). This same trend was also observed in I-SceI-linearized plasmids as the median deletion size was 1097 bp in Ku86-null cells in comparison to 321 bp in wild type cells (Table S2 and Figure S5). When the same analysis was carried out for DNA-PKcs-null (Table S3, S4), XLF-null (Table S5, S6) and LIGIV-null (Tables S7, S8) cell lines less degradation of the DNA ends compared to wild-type cells was observed (Figure S4, S5). In summary, the absence of Ku in human somatic cells carried with it a hyper- resection phenotype that was identical to that observed for Ku- dependent reactions in all other species. A-NHEJ in the absence of Ku in rodent cell lines Given that the hyper-resection phenotype was conserved between human somatic cells and other mammals, we anticipated that the repair phenotypes we have described would also be conserved. To experimentally test this prediction, we utilized two well-characterized hamster cell lines that are defective in Ku86 expression, XR-V9B [68] and sxi-3 [69]. The ‘‘parental’’ cell line from which these two mutant lines were isolated, is the hamster lung cell line, V79-4. Similar to HCT116 cells, the V79-4 cell line was able to convert about 50% of the pEGFP-Pem1-Ad2 linearized plasmid to circularized product regardless of whether it had been linearized with HindIII or I-SceI (Figure 8A). Unlike the human Ku86-null cell line however, the sxi-3 mutant cell line was significantly reduced in its ability to repair, although the XR-V9B line had intermediate activity (Figure 8A). These results were very similar with a published report using another hamster cell line, xrs- 6 and its complemented control, where a 5-fold reduction in repair frequency was observed for the mutant line [37]. Thus, 3 independent hamster Ku86-null cell lines showed a deficit in their repair frequency, which was different from what we have observed in Ku-depleted human somatic cells. To investigate this in more detail, we recovered 14 originally HindIII-linearized plasmids from each of these cell lines and several additional control cell lines and determined the percentage of perfect joins in the products by attempting to re-cleave them with HindIII. Only a single perfectly rejoined plasmid (1/14 = 7%) was recovered from either V79-4 cells or the related ‘‘wild-type’’ Chinese hamster ovary (CHO) line (Figure 8B), consistent with C-NHEJ being the major repair pathway in ‘‘normal’’ hamster somatic cell lines as well. In contrast, both XR-V9B (10/14 = 71.4%) and the sxi-3 (7/ 14 = 50%) cell lines showed heavy usage of microhomology- mediated repair events (Figure 8B). Derivative sxi-3 cell lines, which express either a complementing cDNA (+cDNA#1) or a cDNA expressed in the anti-sense orientation (+AScDNA) have Ku86 Regulates Pathway Choice in Human Cells Figure 8. Microhomology-mediated end joining in hamster Ku86-null cell lines. (A) The indicated cell lines were transfected with HindIII- (Top panels) or I-SceI-(Bottom panels) linearized pEGFP-Pem1- Ad2 together with a supercoiled pCherry plasmid (to monitor transfection efficiency). The number in the top right corner corresponds to the percentage of cells that turned green after 24 hr as a percentage of the cells productively transfected. +cDNA#1 corresponds to a sxi-3 cell line that has been stably complemented with a Ku86 cDNA. (B) HindIII-linearized pEGFP-Pem1-Ad2 plasmids were recovered from the indicated cell lines, propagated through E. coli and then analyzed for retention of a single HindIII restriction site (‘‘perfect rejoining’’) by HindIII restriction enzyme digestion analysis. (C) The indicated cell lines were transfected with the pDVG94 plasmid that had been linearized by Eco47III and EcoRV digestion. After 24 hr, the plasmids were recovered and then analyzed by PCR as described in Figure 7. doi:10.1371/journal.pgen.1000855.g008 been described [70]. These cell lines showed a restoration of C- NHEJ (3/14 = 21.4%) and a lack of restoration (6/14 = 42.9%), respectively. To corroborate these results, the cell lines were also subjected to the independent repair assay using the linearized pDVG94 plasmid. In this instance, both XR-V9B and sxi-3 again showed a much higher usage of A-NHEJ than the parental V79-4 cell line and this could be rescued in two independent cell lines containing a complementing cDNA, but could not be rescued in the cell line containing the cDNA expressed in the anti-sense orientation (Figure 8C). From these experiments, we conclude that although the loss-of-function of Ku86 phenotypes in human cells is PLoS Genetics | www.plosgenetics.org 9 February 2010 | Volume 6 | Issue 2 | e1000855