type (Figure 2). In contrast, the LIGIV+/2 cell line possessed only ,65% the repair capacity of the parental cell line and was reproducibly haploinsufficient (Figure S1 and Figure 2). In no case was a significant difference in repair frequency between the repair of HindIII- or I-SceI-plasmid observed (Figure 2). While the FACS analysis (Figure S1 and Figure 2) measured repair frequency, the repaired plasmids could also be analyzed molecularly. In particular, the HindIII-cleaved substrate contained 4 bp compat- ible overhangs that essentially constituted a stretch of micro- homology (Figure 1B). If these sequences are used to mediate the repair event, they generate a slightly smaller plasmid that now contains a single HindIII restriction enzyme recognition site where formerly there were two. Consequently, the recovered, repaired HindIII-linearized pEGFP-Pem1-Ad2 plasmids were re-digested with HindIII before gel electrophoresis and the frequency of plasmids that had reconstituted a single HindIII site (‘‘perfect joins’’) was determined. Ku70+/2, Ku86+/2 (Table S1), DNA- PKcs+/2 (Table S3), XLF+/2 (Table S5) and LIGIV+/2 (Table S7) cells perfectly rejoined an average of ,37% of the substrates, which was slightly higher than the 23% perfect rejoining observed in wild-type cells (summarized in Figure S2 and Figure 5B). Thus, even though the repair activity was not substantially affected by the loss of one allele of any of the C-NHEJ genes tested, the repair profiles shifted towards microhomology-mediated joining. In several instances, perfectly joined plasmids (as assessed by restriction digest and gel electrophoresis) were sequenced and without exception the existence of the expected single HindIII site was confirmed (data not shown). Lastly, plasmids for those HindIII- linearized plasmids that did not perfectly rejoin and 30 plasmids for all of the I-SceI-linearized plasmids (which can not perfectly rejoin) were sequenced. This analysis demonstrated that the size of the accompanying deletions (Figure S4, S5), the frequency of Ku86 Regulates Pathway Choice in Human Cells microhomology usage, and the frequency of insertions was, with a few interesting exceptions (see the Discussion), comparable to that observed in wild-type cells (Tables S2, S4, S6, S8 and Figure S2, S3). From these experiments, we concluded that the reduction by one allele of most C-NHEJ factors is generally aphenotypic for DNA end joining whereas LIGIV is more haploinsufficient, implying that LIGIV may be a limiting C-NHEJ factor in human somatic cells. This latter conclusion supports a prediction made based on the behavior of purified ligases in vitro [63]. The absence of DNA-PKcs, XLF, and LIGIV greatly reduces DNA repair activity In rodents, cells deficient in any of the C-NHEJ components are generally very deficient in joining virtually all types of DSBs [1]. To test whether C-NHEJ-deficient human cells are also impaired in end joining we repeated the above experiment in DNA-PKcs-, XLF- and LIGIV-null cell lines. In all three cases, the frequency of end joining was greatly reduced (Figure 3). On average, DNA- PKcs- and XLF-null cell lines were diminished by an order-of- magnitude and showed only about 10% the repair activity observed in the parental cell line (Figure 2). The LIGIV-null cell line was always the most profoundly affected cell line and performed end-joining only a few percent above background. The fact that XLF-null cells were reproducibly more active than the LIGIV-null cells is consistent with XLF playing an important, but not essential, role in DNA DSB ligation. We next attempted— as a proof-of-principle—to functionally rescue the XLF-null line to confirm that the loss of end-joining activity was due specifically to the respective targeted knockout mutations in these cell lines. A XLF cDNA was stably introduced via retroviral infection into the XLF-null cell line and a subclone expressing wild-type levels of XLF protein was isolated (data not shown). This cell line showed Figure 2. The impact of C-NHEJ mutations on end joining. Four independent experiments comparable to those depicted in Figure S1 and Figure 4 were performed and the average percent repair relative to wild-type is shown with the standard deviation. doi:10.1371/journal.pgen.1000855.g002 PLoS Genetics | www.plosgenetics.org 4 February 2010 | Volume 6 | Issue 2 | e1000855