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 cell division (43, 87). Further, mice deficient in telomerase experience approximately 5 kb of telomere erosion with every generational intercross to a point at which denuded telomeres are recognized as damaged DNA, participate in end-to-end fusions, and cause progressive degeneration of rapidly dividing tissues (8, 57). Nonhomologous end joining (NHEJ) is a critical pathway responsible for the repair of DNA DSBs in a wide range of organisms (46). In mammals, NHEJ is mediated primarily by the DNA-dependent protein kinase (DNA-PK) complex, comprising the Ku subunits (Ku70 and Ku86), the Lig4-XRCC4 complex, Artemis, the DNA-PK catalytic subunit (DNA-PKcs), and the recently described component XRCC4-like factor (XLF)/Cernunnos (2, 13, 58). Cells and organisms deficient for any one of these proteins exhibit defective DNA DSB repair and, accordingly, are highly sensitive to DNA DSB-inducing agents, such as ionizing radiation. In addition, mammals rely on this repair pathway to generate the immune repertoire of the adaptive immune system, whereby NHEJ proteins function during V(D)J recombination to repair the RAG-mediated DNA DSBs (74). As such, mutations affecting NHEJ are associated with severe immunodeficiency in mice, humans, horses, and dogs (68, 74). The prominent role of the NHEJ pathway in DNA DSB repair also serves to maintain genome integrity, as evidenced by the presence of marked chromosomal rearrangements and the cancer predisposition of various NHEJ mutant mouse strains, particularly in the context of mutant p53 (18, 22, 30, 34, 39, 64, 75, 80, 89, 94). Lastly, deficiencies in various NHEJ components also engender accelerated senescence in cultured cells (23, 30) and segmental aging phenotypes on the whole-animal level (27, 30, 44, 89). Recent work has also described an increasingly complex relationship between NHEJ and telomeres (60). At first glance, telomeres might appear to be ideal substrates for NHEJ activities by nature of their similarity to internal chromosomal DNA DSBs. However, the unique physical structure of the telomere, designated the t-loop (38), appears to prevent the native end of chromosomes from being recognized as aberrant DNA damage (20, 21, 49, 85). Indeed, experiments using mechanisms that disrupt the chromatin structure of telomeres (for example, via expression of dominant negative TRF2) (86) produce telomeres that are substrates for the NHEJ machinery, which subsequently creates interchromosomal fusions that can retain substantial telomere DNA repeats (81). These results corroborate the finding that LIG4-mediated NHEJ creates telomere fusions in Tel1- and Mec1-deficient strains of Saccharomyces cerevisiae (61), as well as in Arabidopsis thaliana, where Ku70 plays at least some role in fusing critically shortened telomeres (45). Further, experiments conducted with NHEJ mutant mice implicate this pathway as a critical mediator of the telomere response when telomeres are eroded beyond their minimum functional length (24, 25). Specifically, in the mTerc−/− mouse model, DNA-PKcs and Ku86 were reported to be required for the creation of the hallmark end-to-end fusions that are devoid of telomere sequences at their junction as a result of telomere attrition (24, 25). Additionally, in the context of these studies, these proteins have been reported to facilitate the induction of the p53-dependent apoptotic response in telomere-eroded tissues, such as the intestine and the gonads (24, 25). Telomere attrition and the presence of telomere signal-free ends is increased in the combined absence of NHEJ and telomerase in mice (24, 25), although it remains unclear whether the accelerated telomere attrition seen in these contexts is a direct result of capping function or is from the increased cellular turnover required to maintain tissue cellularity and function in these genetic backgrounds. In counterpoint to these studies, much work has also conveyed that, rather than functioning solely in response to telomere dysfunction, NHEJ proteins play an important role in normal telomere maintenance (29, 60). In the yeast S. cerevisiae, the Ku subunit binds to the telomerase RNA, perhaps to facilitate normal and de novo telomere access to telomerase (37, 83). Ku mutants in both S. cerevisiae and Schizosaccharomyces pombe have shorter but stable telomeres (4, 12, 69), further implicating the Ku complex in normal telomere maintenance. Mammalian cells also appear to depend on activities of the NHEJ complex for normal telomere maintenance. For instance, cells derived from Ku70, Ku86, Artemis, and DNA-PKcs knockout mice and from DNA-PKcs point mutant (SCID) mice harbor chromosome end-to-end fusions that retain telomere sequences and show increased genomic instability, implicating NHEJ proteins in telomere capping (3, 35, 36, 52, 73, 78). Ku proteins can bind to telomere repeats in vitro (6) and can be localized to telomeric DNA in cells via chromatin immunoprecipitation, and Ku deficiency results in shortened telomeres (19, 51); further, Ku has been reported to bind to telomere repeat binding proteins TRF1 and TRF2 (52, 82). On the other hand, Ku70 deficiency in other species, including chickens and plant species, had either no effect on telomere length (91) or resulted in dramatic telomere lengthening (14, 72). Thus, the impact of NHEJ deficiency on telomere dynamics appears to vary in a manner dependent upon the specific NHEJ mutation, species, and experimental context, and the precise roles of these factors in telomere length regulation continue to be areas of active investigation (19, 24, 35, 36, 42, 78). Given the range of functions and complex phenotypes associated with the core NHEJ components in telomere biology, we sought to understand the genetic interactions of NHEJ and telomeres in the response to physiological telomere attrition in vivo by generating and characterizing mTerc−/− mice and cells that are also null for DNA-PKcs or Lig4. Our results indicate that classical NHEJ is dispensable for the telomere erosion-dependent chromosome fusions and, further, that DNA-PKcs has no impact on the apoptosis response induced by telomere erosion in multiple organ compartments. The results of this study stand in sharp contrast to the proposed roles attributed to the NHEJ complex, including DNA-PKcs, in the telomerase-deficient mouse (26). Furthermore, the strict requirement of NHEJ in the repair of dysfunctional telomeres generated by acute experimental disruption of telomere binding proteins (80) versus the dispensability of NHEJ in the fusion of dysfunctional telomeres arising in the setting of telomerase deficiency and telomere erosion indicates that physiological telomere attrition can engage non-NHEJ pathways to resolve dysfunctional telomeres.