The Ku heterodimer performs separable activities at double-strand breaks and chromosome termini.

Telomeres are essential structures that constitute the ends of linear chromosomes. In the majority of eukaryotes studied, telomeric DNA consists of tandem arrays of simple G-rich sequence which are maintained by the enzyme telomerase (39). The G-rich strand extends 5′ to 3′ toward the chromosome end, terminating in a single-stranded 3′ overhang. The assembly of both duplex and single-strand telomeric DNA binding proteins onto telomeric DNA gives rise to a nonnucleosomal structure. A primary function of this telomeric nucleoprotein complex is to prevent the natural chromosome ends from being subject to DNA repair and recombination activities, thereby preventing end-to-end fusions and unregulated resection of terminal DNA sequences (10). In contrast, DNA ends created by double-strand breaks (DSBs) are targets for repair activities, thus averting the toxic consequences to the genome if a DSB is left unrepaired (19). In the nonhomologous end-joining (NHEJ) pathway of DSB repair, broken ends with little or no sequence homology are joined with minimal, if any, sequence loss (32). In both Saccharomyces cerevisiae and higher eukaryotes, the Ku heterodimer is required for this process to occur both accurately and efficiently (6, 40). Consisting of 70- and 80-kDa subunits, this abundant heterodimeric complex binds with high affinity to DNA ends in a sequence-independent fashion and to a variety of terminal structures, such as blunt ends and 3′ overhangs (15). As predicted by its known in vitro binding properties, Ku localizes to DSBs in vivo (38). Biochemical studies suggest that Ku acts at DSBs as a bridging complex, an alignment factor, and a recruitment factor in the end-fusing process (18, 27, 42, 46, 51). Additionally, in yeast cells, there is enhanced degradation of broken chromosomes in the absence of Ku, suggesting a role for Ku in the protection of DNA ends from nucleolytic processing (29). Unexpectedly, the Ku heterodimer also localizes to telomeres in both yeast and mammalian cells, and loss of Ku function results in dysfunctional telomeres across species (20, 23). Disruption of Ku activity in budding yeast cells, due to mutations in either of the Ku subunits (encoded by YKU70 and YKU80), results in a number of telomere-related phenotypes, suggesting that Ku has multiple activities at chromosome ends. First, Ku is required for telomere length regulation (50), an activity that is mediated through a 48-nucleotide stem-loop structure of TLC1, the RNA subunit of yeast telomerase (48, 55). In addition, Ku contributes to yeast telomeric chromatin structure, as expression of a telomeric reporter gene is altered in strains that lack either YKU70 or YKU80 (5, 28, 43). The Ku heterodimer also makes critical contributions to the protection of chromosome ends. In budding yeast cells, Ku protects telomeres from homologous recombination (14). In addition, regulation of the terminal G-rich single-strand overhang is lost in Ku-deficient strains (20, 49) due to unregulated resection of the telomeric C-rich strand (37). Furthermore, budding yeast strains deficient in both telomerase (e.g., due to loss of EST2, the telomerase catalytic subunit, or EST1, a telomerase regulatory subunit) and Ku display a greatly accelerated senescence progression compared to that exhibited by a telomerase-deficient strain (20, 43, 49). Similarly, rapid loss of telomeric DNA and decreased survival have also been observed in fission yeast strains that lack both Ku and telomerase (4). The inviability displayed by these double-mutant strains is thought to be due to the simultaneous inability to both replicate and protect chromosome ends (A. Bertuch and V. Lundblad, submitted for publication). Ku has also been implicated in chromosome end protection in mouse cells, based on the increase in chromosomal end-to-end fusions that are observed in the absence of Ku function (3, 9, 23, 53). Moreover, the degree of telomeric dysfunction observed in Ku-deficient cells, as measured by end-to-end fusions, is comparable to that observed in late-generation telomerase-deficient mice (16). Insight into Ku's function at a molecular level has come from the determination of the crystal structure of the human Ku heterodimer, both alone and in complex with a DNA substrate (61). These structures reveal that the Ku70 and Ku80 protein subunits share a three-domain topology, with each comprised of an N-terminal α/β domain, a central β-barrel domain and a C-terminal arm. The associated subunits give rise to a quasi-symmetric basket-like molecule with a narrow preformed ring, which cradles and encircles the DNA. The N-terminal α/β domains, which lie at the periphery of the heterodimer, contribute minimally to heterodimerization or DNA binding and are potentially involved in interactions with other proteins. Together, the β-barrel domains form the DNA binding surface and the ring-like structure through which the duplex DNA is threaded. Last, the helical C-terminal arms of each subunit are positioned to contact the β-barrel of the opposite subunit, thereby contributing to heterodimerization. The structure of the heterodimer-DNA complex can be readily incorporated with current models of Ku function in NHEJ. For example, the ring-like structure is consistent with its requirement for DNA ends, and the absence of specific contacts with DNA bases explains its sequence-independent mode of binding. The positioning of the DNA helix fits well with a role for Ku as an alignment factor, yet the ends are left accessible to end processing events. How the Ku heterodimer dissociates from a DNA end once end joining has occurred and whether translocation occurs in vivo, as has been observed in vitro (11), are questions that have not yet been resolved. The dual requirement at DSBs, where Ku promotes end-to-end fusions, and at telomeres, which are specifically protected from end joining, presents a paradox. What prevents Ku from mediating the same kinds of events at telomeres that it promotes at DSBs? A possible resolution of this paradox is a model in which Ku performs different activities at these two different classes of DNA ends. For example, telomere-specific proteins could modulate an activity performed by Ku when it is associated with chromosome ends that is precluded from occurring at DSBs. To test this model, we asked whether mutations in YKU80 that separate its DNA repair function from its telomere function could be recovered. We report here the identification of mutations, called yku80tel alleles, which result in striking defects in telomere maintenance but are still proficient for DNA repair. This supports a model in which Ku performs different activities at these two different types of ends. Furthermore, the yku80tel mutations differentially impact telomere length regulation, telomere end protection, and telomeric silencing, suggesting that Ku has separable functions at the telomere as well. Based on sequence alignment and homology modeling with the human Ku structure, four of the mutated residues map to within the N-terminal α/β domain. Because this domain has been proposed to mediate interactions between Ku and other factors (61), we suggest that this is a region that is critical for telomere-specific interactions.