mimicked in the Ku86-null hamster cells by the heavy reliance on A-NHEJ, the ability to de-repress A-NHEJ—as scored by these extrachromosomal assays—is not conserved. Discussion We have utilized rAAV knockout technology to construct a powerful reagent: a series of isogenic human cell lines that are defective for genes required for the C-NHEJ-mediated branch of DNA DSB repair. We used these cell lines along with several informative reporter systems to demonstrate that wild-type human cells vastly prefer to utilize C-NHEJ over A-NHEJ for end joining reactions. Unexpectedly, the absence of the proximal C-NHEJ factor Ku, resulted in cells that still carried out robust levels of end joining, suggesting that Ku normally suppresses other end joining pathways. This model was supported by the construction of double mutant cell lines, which demonstrated that the reduction of Ku in a cell that was incapable of carrying out C-NHEJ still resulted in high levels of end joining. Thus, these studies demonstrate that Ku is the critical regulator for determining pathway choice in human somatic cells. Ku, the ‘‘mother’’ of all DSB repair inhibitors? Ku is a heavily researched DNA repair factor and the majority of studies rightfully concentrate on some aspect of Ku’s ability to positively facilitate the myriad of repair and recombination reactions that require C-NHEJ. In this and related studies, we have recently documented that Ku has an additional and hitherto underappreciated function—it is a powerful inhibitor for all the other DNA DSB repair pathways. Specifically, we have demon- strated that Ku is an essential repressor of HR-mediated aberrant telomere recombination. In the absence of Ku, the HR apparatus can apparently gain access to the telomeric ends and generate lethal telomeric shortening [43,44]. Thus, Ku can inhibit HR specifically at telomeres. Moreover, in a study that characterized generalized rAAV-mediated gene targeting—a process that requires HR—for loci scattered throughout the genome the absence of Ku resulted in a ,10-fold increase in correct gene targeting [42]. Importantly, the increase in correct gene targeting came at no expense to random integrations. These data strongly suggested that the reduction of Ku in human somatic cells de- repressed HR enough to facilitate much higher levels of gene targeting while simultaneously allowing other repair pathways to carry out random integrations at wild-type levels. This conclusion is completely supported by the data provided in this present study. Thus, here we have documented that in the absence of Ku, A- NHEJ is greatly up-regulated. Together, these and other [5,37] studies have revealed that Ku can inhibit HR at telomeres, and it can inhibit HR and A-NHEJ throughout the genome. Moreover, this work and the work of Fattah et al. [42] make the strong prediction that the random rAAV integrations observed in Ku- deficient cells are mediated by A-NHEJ. Is the inhibitory activity of Ku conserved? Although we observed the reliance on A-NHEJ in Ku-deficient hamster cell lines, we did not observe the strong de-repression of A-NHEJ activity that we had observed for human somatic cells (Figure 8A). An identical lack of de-repression using the same assay system was reported in a set of independent hamster Ku86 mutant cell lines [37] and a similar conclusion had previously been reached using extracts derived from these hamster mutants in vitro [16]. However, this de-repression has been observed in rodents by a number of investigators, predominately in mouse MEF or ES cell lines [15,19,21,64,71], but also in hamster cell lines [17,18,20]. Ku86 Regulates Pathway Choice in Human Cells The difference between these studies is that ours and the ones where de-repression was not observed were performed using extrachromosomal reporters and those where de-repression was observed were performed with chromosomally integrated reporter constructs. Thus, it appears in mammals as if the presence of Ku is more critical for chromosomal DSBs (which are of course the most biologically relevant substrate) and their repair than for extra- chromosomal DSB repair. The basis for this distinction is not known, but it is very likely linked to Ku’s ability to affect chromatin structure and the impact (direct or indirect) that this may have on repair processes. Consistent with this view, Ku separation-of-function mutations, which specifically alter Ku’s heterochromatin activities, have been demonstrated to affect yeast Ku’s ability to modulate telomere recombination [72]. Together, these results suggest that the difference between humans and rodents is not one of kind, but one of degree. Thus, virtually all of the loss-of-function phenotypes that have been described for human Ku can be observed in rodent Ku mutant lines, although the effects are generally—and with profound consequences— ameliorated. How does Ku orchestrate all this inhibition? Many models can be envisioned for how Ku suppresses A- NHEJ. One possibility is that Ku, via direct protein:protein interaction, sequesters a key A-NHEJ factor from performing its function. In a Ku-deficient cell, this factor would be free to facilitate A-NHEJ. A good candidate for such a putative factor exists. Thus, a bevy of independent laboratories have demonstrat- ed that PARP-1 interacts with Ku [73–77]. And a PARP-1 interaction domain has been defined in the Ku70 subunit at AA243–261 [76]. This model predicts that a cell expressing a Ku70 incapable of interacting with PARP-1 (e.g., mutated at residues AA243–261) would phenocopy the Ku loss-of-function mutations and we are attempting to construct such a cell line. A second, and in our minds, likelier possibility, is that Ku controls A- NHEJ by regulating access to the substrate; namely, a dsDNA end. We prefer this model because not only does Ku repress A-NHEJ but it also represses HR at internal loci [42] and at telomeres [43,44,72]. While it is possible that Ku mediates all of this repression by physically binding to and inhibiting/sequestering a different protein or proteins for each reaction, it seems simpler if Ku simultaneously regulates all three processes by regulating access to the substrate for all of these pathways: double-stranded DNA ends. Specifically, we propose that in order to be channeled into a particular pathway (HR, A-NHEJ or C-NHEJ), that pathway’s DNA binding factor (probably RAD52, PARP-1 and Ku, respectively) needs to bind onto the ends of the break and subsequently recruit their pathway’s associated factors. We posit that Ku generally gets to the ends of a dsDNA break faster and/or with higher affinity than RAD52 or PARP-1 and once there it blocks their access, such that repair is fated to occur by C-NHEJ. This model is by no means novel and has been proposed by several investigators and was broached at least a decade ago [78], although it still remains largely untested. In this regard, a cell line that expressed a double-stranded DNA end binding defective Ku mutant would be predicted to be incapable of repressing either HR or A-NHEJ. Lastly, this model, in particular, could explain the differences between mice and humans. In mice, the levels of Ku/ DNA-PK are much lower than they are in humans and consequently there might be a ‘‘fair fight’’ between Ku, RAD52 and PARP-1 over who gets to a broken end. In contrast, in human cells where the levels of Ku/DNA-PK are about 50-fold higher [11,79], Ku has become the ‘‘bully’’ and essentially dominates pathway choice. PLoS Genetics | www.plosgenetics.org 10 February 2010 | Volume 6 | Issue 2 | e1000855