Ku's essential role in keeping telomeres intact.

T elomeres, the specialized nucleoprotein structures present at the ends of linear chromosomes, function to prevent natural chromosomal termini from activating the DNA damage response and becoming substrates for inappropriate DNA repair. Telomeres are organized into lariat-like structures known as tloops, which are formed by the invasion of the terminal G-rich 3 telomeric overhang into the proximal duplex telomeric tract on the same chromosome (1) (Fig. 1). T-loops have been proposed to be a crucial means by which the telomeric end is hidden from DNA double strand break (DSB) repair pathways, with nonhomologous end joining (NHEJ) being the dominant mechanism of DSB repair in mammalian cells. Consequently, when telomeres become dysfunctional, and presumably no longer able to form tloops, they can be engaged in NHEJ, giving rise to chromosome end-to-end fusions (2). Paradoxically, the Ku70/ Ku86 heterodimer, a central component of NHEJ, is important at functional telomeres. There, instead of mediating NHEJ, Ku has been shown to contribute to various aspects of telomere structure and function. For example, Ku has been found to protect telomeres from inappropriate degradation and interchromosomal recombination and contribute to the tethering of telomeres to the nuclear periphery and the regulation of telomerase (3). Although Ku has a conserved role in NHEJ and appears to have at least one or more roles at the telomere across species, only in humans has it been shown to be an essential protein (4). The reason has been unknown, but it has been speculated that Ku’s essential function lies in its telomeric, not NHEJ, role. In this issue of PNAS, Wang et al. (5) have not only confirmed this speculation but have revealed that Ku’s essential role in human cells is to prevent dramatic telomere loss. In their article, Wang et al. (5) demonstrated that the cell death that resulted from the conditional knockout of Ku86 in a human somatic cell line was associated with massive telomere loss. Telomere FISH showed that, on average, a striking 66% of chromosome ends were devoid of telomere signal after depletion of Ku. These so-called signalfree ends were associated with the appearance of extrachromosomal telomere circles (t-circles), which suggests that the telomere loss was the result of telomere rapid deletion, a previously described phenomenon that results in telomere shortening caused by intrachromosomal recombination (6). Additionally, 50% of Ku-deficient cells accumulated -H2AX foci, indicative of DNA ends being recognized as DSBs. This number is much greater than what was observed in a cell line that lacked DNA-PKcs, a protein that associates with DNA-bound Ku and is crucial for NHEJ-mediated repair. Moreover, the DNA-PKcs / cell line was viable and did not exhibit massive telomere loss (7). The results taken together, therefore, strongly suggest that the essential function of Ku86 in human cells is not to perform NHEJ, but rather to prevent the elimination of telomeres and the formation of t-circles. A fair amount is known about t-circles already, because they have been observed in a number of settings. In many cases, t-circles are thought to arise from recombinational excision of t-loops (8). This mechanism makes sense when one considers that the base of the t-loop, known as a displacement loop (d-loop), resembles a Holliday junction (HJ) intermediate (Fig. 1). In contrast to what occurs in homologous recombination, branch migration and HJ resolution are suppressed at telomeric d-loops because these activities would result in the excision of a t-loop and a shortened telomere. The N-terminal basic GAR domain of TRF2, a component of the telomere maintenance complex shelterin, appears to play a major role in this suppression. This domain is required for TRF2 inhibition of resolvase cleavage of telomeric HJs in vitro and both mouse and human cells expressing high levels of TRF2 B, which lacks the domain, exhibit high levels of t-circles (9, 10). Additional factors implicated in t-circle formation in response to TRF2 B expression include XRCC3, which is associated with HJ resolvase activity in mammalian cells; Nbs1, a component of the Mre11 complex, which is involved in recombinational repair; and WRN, an helicase/ exonuclease, which interacts directly with TRF2 (9, 11). Of interest now is determining the mechanism by which Ku functions to prevent telomere loss and t-circle formation, and there are suggestions it may prove to be novel. Wang et al. (5) discuss that Ku has been shown to interact with both TRF2 (12) and WRN (13), supporting the notion that it might mediate its effects through one of these proteins. However, TRF2 deficiency or WRN dysfunction results in preferential loss of leadingor lagging-strand telomeres, respectively. In contrast, Wang et al. found that the telomere loss exhibited in Ku86-deficient cells was random. This result suggests that Ku prevents t-circle formation via either a combined disturbance in TRF2 and WRN function or a distinct mechanism like inhibiting directly or recruiting a repressor of recombination (Fig. 1 A). That Ku might function through a distinct pathway is also supported by findings in Arabidopsis, in which Ku was