The repair of DNA double-strand breaks (DSBs) is important for cellular survival, the maintenance of genomic integrity, and the prevention of tumorigenesis. DSBs can be caused by exposure to exogenous agents, including ionizing radiation and chemotherapeutic agents, and be generated by endogenous mechanisms, such as variable (diversity) joining [V(D)J] and class-switch recombination processes required for the maturation of B and T lymphocytes. Moreover, the ends of linear chromosomes present cells with naturally occurring DSBs i.e., telomeres – and these must be regulated to ensure the stable maintenance of the genome.

Because of the importance of DNA repair, immune function, and genomic stability for organismal well-being, at least 2 mechanisms for the repair of DSBs have evolved: homologous recombination (HR) and nonhomologous end-joining (NHEJ). In lower eukaryotes, HR, which requires regions of homology between the donor and the recipient DNAs, is the major pathway for general DNA DSB repair. In higher eukaryotes, HR is also important for meiosis, sister chromatid exchange, and the repair of stalled replication forks.

Despite the importance of HR, the process of NHEJ, in which 2 DNA ends are joined together regardless of their DNA sequence homology, nonetheless predominates in mammals. The most upstream, and quite probably the most important NHEJ factor, is the Ku complex.

Ku is conserved from bacteria to humans. In bacteria, Ku exists as a homodimer, but in all other species it exists as 2 independent subunits, Ku70 and Ku86, that tightly heterodimerize. Importantly, in all organisms examined – with one glaring exception – mutations of either Ku subunit result in the expected deficits in DNA DSB repair and recombination. Intriguingly, humans are unique in that Ku has apparently evolved into an essential gene. This hypothesis is supported by the lack of documentation for even a single patient with a mutation in either Ku subunit.

The absence of human Ku patients stands in contrast to some of the more downstream NHEJ factors, such as DNA-PKcs, Artemis, Cernunnos/XLF, and DNA LIGIV, for whom mutations in human patients have been described. Thus, it appears as if Ku – but not NHEJ – is essential. The bias that Ku may be essential in humans is also supported by the demonstration that the targeted disruption of both alleles of either Ku70 or Ku86 in human somatic cells was lethal. And although it was impossible to discern the mechanism of cell death in these studies, the fact that the inactivation of one allele of either Ku70 or Ku86 by gene targeting or the reduction of Ku86 levels byRNAi resulted in telomere shortening suggested by extrapolation that the lethal event in a completely Ku-deficient human cell might be due to aberrant telomere maintenance.

Why human Ku-deficient cells should suffer from lethal telomere maintenance events when this is not observed in the cells/organisms of other Ku null species has been difficult to envision? What is clear, however, is that telomere maintenance is a species-idiosyncratic process. Ku and Ku mutants exemplify this. Thus, mutation of either Ku subunit in most species results in telomere defects.

Despite this uniformity, however, there are some glaring incongruities with the phenotypes of the respective mutants. Thus, Saccharomyces cerevisiae Ku mutants show some telomere shortening and a high temperature lethality associated with defective telomere maintenance. In contrast, Arabidopsis thaliana Ku mutants show massive telomeric expansions.

Different yet from all these are chicken DT40 cells, where no telomere defects or only slight expansions have been reported.

Finally, the mouse literature is conflicted, with slight telomeric expansions or significant telomere shortening being reported for similar mouse strains.

Altogether, the diametrically opposed results generated from different model organisms combined with the apparent lethality of Ku-defective human cells has resulted in a conundrum concerning the impact of Ku mutations on human telomere maintenance. To scientifically address this issue, scientists constructed a human cell line that is conditionally null for Ku86. Upon expression of the Cre recombinase, the only functional allele of Ku86 is lost from the genome. The resulting cells are not viable, consistent with earlier observations.

Importantly, it now can be demonstrated that cell death is associated with a telomere loss so rapid and extensive that it has no parallels in the mammalian literature. The telomere loss occurs nearly en masse in the form of extrachromosomal t-circles, and this is consistent with human Ku86 being an essential regulator for the suppression of rapid telomere loss.