Author Summary Humans utilize at least two major pathways to repair DNA double-strand breaks (DSBs): homologous recombination (HR) and non-homologous end joining (NHEJ), and there are at least two genetically discrete sub-pathways of NHEJ: classical-NHEJ (C-NHEJ) and alternative-NHEJ (A-NHEJ). Since the products generated by each of these three repair (sub)pathways differ substantially from one another, it is biologically critical that certain DSBs are repaired by certain DSB repair pathways. How this pathway choice is made in human cells was unclear. In this study, knockout human cell lines that are defective in core C-NHEJ factors were generated. These cell lines are by-and-large extreme- ly deficient in DSB repair, proving that C-NHEJ is the major DSB repair pathway in human cells. Unexpectedly, cell lines reduced for the C-NHEJ factors Ku70 or Ku86, carried out proficient DSB repair because of hyperactive A-NHEJ. In published work we have also demonstrated that Ku suppresses HR throughout the genome and at telomeres. Collectively, these data imply that Ku ensures that C-NHEJ is the major DSB repair pathway by two mechanisms: i) enabling C-NHEJ and ii) by actively suppressing HR and A- NHEJ. Thus, Ku is the critical regulator of pathway choice in human somatic cells. PKcs. DNA-PKcs, in turn, phosphorylates and activates the nuclease Artemis, which facilitates ‘‘cleaning up’’ of the ends. As a final step, ligation of the broken ends is catalyzed by the trimeric LIGIV complex, which consists of the catalytic core, DNA LIGIV, and its two accessory factors, XLF and XRCC4. In contrast to C-NHEJ, the mechanism, the regulation and the factors involved in A-NHEJ remain elusive. Mechanistically, it is believed that during A-NHEJ both broken ends are resected 59-to- 39 on one strand to generate 39-single-stranded overhangs containing regions of microhomology (generally a few nucleotides), which are then used to mediate the repair event. Because of this reaction pathway, deletion of the sequences between the microhomologies occurs as does deletion of one of the blocks of (micro)homology. Moreover, the remaining block of microhomol- ogy always resides at the precise site of repair and can be used as a landmark to define such repair events [6,9]. A-NHEJ was not thought to be a very robust nor particularly important DSB repair pathway because it could usually only be detected in the absence of C-NHEJ. Indeed, one of the first descriptions of A-NHEJ came with the observation that the few NHEJ DSB repair events that could be detected in Ku86-deficient budding yeast occurred between short direct repeats [7]. Since then, there have similar reports in fission yeast [13], frogs [14] and several mammalian systems [15–22] including humans [23]. The significance of—and parallel interest in—A-NHEJ increased with the demonstration that A-NHEJ could substitute at reasonable levels for C-NHEJ during DNA DSB repair events in murine lymphoid class switch recombination [24,25] and during certain types of aberrant V(D)J recombination reactions [26,27]. More- over, A-NHEJ has been implicated in the generation of large deletions and other genomic rearrangements in murine cells [28–30]. Similarly, microhomology has been found at the recombination junctions of radiation-induced genomic rearrange- ments [31,32] implying that even radiation-induced DSBs can be repaired by A-NHEJ. Lastly, microhomologies are frequently detected at breakpoints for chromosomal deletions and transloca- tions in human cancer cells [33,34]. These observations have propelled many laboratories to identify the factors required for A- Ku86 Regulates Pathway Choice in Human Cells NHEJ. These studies have implicated poly (ADP-ribose) polymer- ase-1 (PARP-1), X-ray cross complementing 1 (XRCC1), DNA ligase III (LIGIII), polynucleotide kinase (PNK), Flap endonucle- ase 1 (Fen-1) [5,14,35–37] and most recently the Mre11:R- ad50:Nbs1 (MRN) complex (reviewed by [38]) but it is clear that additional factors await identification. One of the most compelling questions in the DSB repair field is how pathway choice is determined. That is, once a chromosome breaks, how does the cell determine whether HR, C-NHEJ or A- NHEJ will mediate its repair? Since each of these repair pathways generates a discretely distinct product, the answer to this question is biologically important. Several laboratories have suggested that the relative abundance of factors, binding affinities for DNA ends, cell type specificity and/or cell cycle phases may impact upon this decision {reviewed in [39]}. These issues are complicated even more in human somatic cells where the impact of loss-of-function mutations on some of the C-NHEJ genes has distinctly different phenotypes than are observed in other mammals. In particular, Ku70 and Ku86 have evolved an essential telomere maintenance function that does not seem to be evident in any other mammalian studied to date [40–43]. Interestingly, Ku seems to exert this function by repressing the HR-mediated disassembly of telomeres [44] suggesting that pathway choice is critical for naturally occurring double-stranded DNA ends as well as broken ones. To begin to experimentally address some of these issues we have generated a series of human somatic cell lines genetically engineered using recombinant adeno-associated virus (rAAV)- mediated gene targeting [45–47] to contain reduced levels of the C-NHEJ factors Ku70, Ku86, DNA-PKcs, XLF and LIGIV. We hypothesized that in the presence of reduced or no C-NHEJ activity the frequency and regulation of A-NHEJ in human cells could be assessed. To this end we utilized two in vivo plasmid assays that have been employed to study end joining in mammalian cells [26,37,48] to demonstrate that mutations in DNA-PKcs, XLF or LIGIV resulted in a severe reduction in the frequency of productive DNA DSB repair. The small number of repair events that did occur in these cell lines were hallmarked by the heavy usage of microhomology. Thus, these studies confirmed that C- NHEJ is the dominant NHEJ pathway operative inside human somatic cells and that in its absence small amounts of A-NHEJ can be detected. Very surprisingly, and in stark contrast to the results with DNA-PKcs, XLF and LIGIV-null cell lines, DNA DSB repair activity was actually slightly elevated in Ku86 conditionally-null cell lines. These repair events appeared, once again, to be heavily biased towards microhomology-mediated repair. This result suggested that Ku86 actively suppresses A-NHEJ in human somatic cells. This hypothesis was confirmed by using molecular and genetic approaches to reduce the levels of Ku70 in cell lines that were for either DNA-PKcs or LIGIV and which resulted in cell lines that had regained a DNA DSB repair activity that was mediated by microhomology. Together, these studies demonstrate that Ku (Ku70 and Ku86) is the critical regulator of pathway choice in human somatic cells. Results Strategy and cell lines To elucidate the role of C-NHEJ factors in DNA DSB repair, we made use of an extrachromosomal reporter assay system {[37,48]; Figure 1}. This assay permits, in addition to the generation of defined DSBs, a detailed follow up of the repair of the reporter plasmid. In this assay, end joining is measured by the reconstitution of green fluorescent protein (GFP) expression [48]. The reporter pEGFP-Pem1-Ad2 consists of the GFP gene PLoS Genetics | www.plosgenetics.org 2 February 2010 | Volume 6 | Issue 2 | e1000855