Is there evidence for yet another sub-pathway of NHEJ? In this study we have interrogated our mutant cell lines with two structurally similar, but fundamentally different, types of DNA ends. In one of them (HindIII-linearized pEGFP-Pem1-Ad2) a region of pre-existing microhomology was presented to the cell. In the other (I-SceI-linearized pEGFP-Pem1-Ad2 and linearized pDVG94) some processing by the cell was required to reveal the microhomology. Microhomology-mediated end joining of the HindIII-linearized pEGFP-Pem1-Ad2 plasmid could be detected in the wild-type parental cells (Figure 5 and Tables S1, S3, S5, S7). This perfect end joining increased in Ku heterozygotes and became the dominant reaction product in Ku- reduced/null cell lines. These results can be most simply interpreted if Ku inhibits A-NHEJ and as the level of Ku is reduced the levels of A-NHEJ reciprocally rise. The data generated using the two repair substrates that required processing suggests that this model is, however, over-simplified. Thus, as the level of Ku was reduced in the various cell lines the frequency of microhomology-mediated end joining increased with I-SceI-linearized pEGFP-Pem1-Ad2 and linearized pDVG94, but so did other end joining activities. This was most evident in the experiments using pDVG94 where ,55% of the repaired plasmids in a Ku-null cell did not use microhomology to repair the plasmid (Figure 7C). Sequencing of these events and those derived from I-SceI-linearized pEGFP- Pem1-Ad2 did not reveal any novel repair signatures and most events looked indistinguishable from typical C-NHEJ products (Table S2). Together, these studies suggest that there may be at least one additional NHEJ pathway that is distinguishable from C- NHEJ and A-NHEJ by its lack of requirement for Ku and its lack of microhomology use, respectively. Needless to say, these products could also be accounted for by A-NHEJ if the pathway does not have an absolute requirement for microhomology. The construction of humans cell lines that are doubly defective for C- NHEJ and A-NHEJ should genetically address this issue. The power of rAAV-mediated human somatic cell genetics Advances in the DNA DSB repair field have come predomi- nately from studies on yeast and genetically modified mice. There are instances, C-NHEJ foremost among them, however, where the phenotypes of yeast and mice mutants do not accurately recapitulate the corresponding phenotypes observed in humans. Since ultimately we wish to apply what we have learned in model systems to the study of humans in the clinic, a potentially more appropriate model system is the use of human somatic cells in culture. There are, of course, attendant limitations to using human cells in culture and the requisite caution needs to be taken in extrapolating cell culture results to patients in the clinic. It is, however, also reasonable to expect that the physiology of human cells in culture may reflect more accurately the basic biochemical process of human patients than, say, rodent cell in vivo might. The strength of the rodent system stems predominately from the ability to make targeted alterations of individual genes using the technology of HR [80]. This technology exists for human somatic cells as well [46,47,81]. Overall, at least 77 different genes have been functionally inactivated in a total of 45 different human somatic cell lines {[47] and unpublished data}. To our knowledge, however, this is one of the first reports of the systematic inactivation of a large number of genes involved in a single pathway. Importantly, we have shown that it is possible to make simple knockouts, conditional knockouts and double mutant human somatic cell lines with relative ease. The general utility of rAAV-mediated gene targeting may thus be of interest for Ku86 Regulates Pathway Choice in Human Cells investigators working on biological problems that cannot be adequately modeled in, for example, the mouse. Materials and Methods Cell culture The human wild-type HCT116 cell line and its derivatives were cultured in McCoy’s 5A medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin in a humidified incubator with 5% CO2 at 37uC. Cell lines derived from correct gene targeting were propagated under G418 (1 mg/ml) selection. Cell lines carrying exogenous cDNA expression vectors (either XLF or DNA-PKcs) were grown in 2 mg/ml of puromycin. Cell lines The wild-type human HCT116 cell line was obtained from the ATCC. The derivative Ku70+/2 [50], Ku86+/2 [40], Ku86flox/2 [44], DNA-PKcs+/2 and DNA-PKcs2/2 [51] cell lines have been described. Derivatives of Ku70+/2 cells treated with Ku70 RNAi (SMARTPool oligonucleotides; Dharmacon) or stably expressing shRNA vectors directed against Ku70 have also been described [42]. The XLF+/2 and XLF2/2 (Fattah et al., manuscript in preparation) and the LIGIV+/2 and LIGIV2/2 (Oh et al., manuscript in preparation) cell lines were generated by rAAV gene targeting. Similarly, compound mutant cell lines (e.g., Ku70+/2: LIGIV2/2) were generated using the rAAV targeting technology described elsewhere [47]. Treatment of Ku86flox/2 cells with Cre To generate Ku86-null cells, the Ku86flox/2 cells were plated onto a 6-well plate at a density of 56104 cells per well and allowed to attach for 18 hr. Adenoviral infection was carried out by adding 2 ml of fresh media containing 56108 virus particles of either a control (AdCMV) or experimental (AdCre) adenoviral stock to each well [44]. After 4 days (96 hr) of incubation the cells were re- plated into 6-well plates and allowed to incubate for another 24 hr before the cells were transfected with linearized NHEJ substrates (see below). Flow cytometry was then carried out after an additional incubation for 24 hr. The end-joining assay, transfection, and FACS analyses The in vivo end-joining reporter plasmid pEGFP-Pem1-Ad2 (Figure 1) has been described [37,48]. Prior to transfection, the pEGFP-Pem1-Ad2 plasmid was digested with HindIII or I-SceI (NEB) for 8 to 12 hr to generate different types of DNA ends. A pCherry plasmid (Clontech) was co-transfected with linearized pEGFP-Pem1-Ad2 as a control of transfection efficiency. The cell line under analysis was subcultured a day before transfection and was ,60 to 70% confluent for transfection. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to manufacture’s instructions. Green (EGFP) and red (Cherry) fluorescence was measured by fluorescence-activated flow cytom- etry (FACS) 24 hr later [37]. For FACS analysis cells were harvested, washed in 1X PBS and fixed using 2% paraformalde- hyde. FACS analysis was performed on a FACSCalibur instrument (BD Biosciences). For the HCT116 cell line a red- versus-green standard curve was derived with varying amount of cherry and green plasmids to avoid measurements near the plateau region. The values of repaired events are reported as a ratio of cells that were double positive for red and green fluorescence over total cells that are only positive for red fluorescence. This ratio normalizes the repair events to the transfection controls. The values for all the mutants are reported as a percent repair of wild-type cells. PLoS Genetics | www.plosgenetics.org 11 February 2010 | Volume 6 | Issue 2 | e1000855