Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerasedeficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc ؊/؊ DNA-PKcs ؊/؊ cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)-an additional crucial NHEJ component-was also permissive for chromosome fusions in mTerc ؊/؊ cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNAPKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc ؊/؊ tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.Telomeres are key structural elements for the protection and maintenance of linear chromosomes that are comprised of a series of repetitive DNA sequence elements (TTAGGG in humans) and an array of sequence-specific and -nonspecific DNA binding proteins that create a higher-order chromatin structure (21). Telomeres serve various cellular functions, including the prevention of recognition of the linear end as a DNA double-strand break (DSB) (20,49,85); the modulation of local chromatin structure, which influences the expression of those genes proximal to chromosome ends (5, 66, 67); and the regulation of telomere maintenance enzymes and their accessibility (21). The last function is crucial, given the end replication problem that plagues conventional DNA synthesis (65,90). Telomere sequences can be added by telomerase, a specialized reverse transcriptase that adds telomere repeat DNA to the ends of chromosomes, thereby restoring sequences lost after RNA primer removal and end processing following DNA replication (7,53). Human cells in culture that divide in the absence of telomerase experience telomere attrition on the order of 50 to 100 bp per ce...