The DNA end-binding protein Ku associates with human telomeres primarily via protein-protein interactions

The Ku heterodimer (Ku70/Ku80) binds DNA ends with high affinity but without sequence specificity and, upon binding ends created by double-stranded breaks (DSBs), initiates canonical nonhomologous end-joining (c-NHEJ). Ku also localizes to functional telomeres where its c-NHEJ activity is inhibited. Interestingly, Ku has been co-opted at telomeres across species, where it performs varied telomeric functions. In humans, Ku is essential for its role in telomere maintenance, but how it associates with human telomeres is not known. Analysis of Kus telomere association in different populations of cen3tel cells, which had a wide range of average telomere lengths, supported Kus localization at human telomeres primarily via protein-protein interaction. We also found that the Ku70 and Ku80 5 helices, which are on opposing sides of the heterodimer and were previously implicated in Saccharomyces cerevisiae Kus NHEJ and telomeric functions, respectively, participated in Kus telomere association in human cells. While the Ku70 5 mutant showed increased interaction with TRF2, the Ku80 5 mutant was not impacted for TRF2 association. Interestingly, residues altered to impair Kus DNA end-binding function were also involved in TRF2 interaction and telomere association. Overall, our results suggest protein-protein interactions as the primary mode by which Ku associates with human telomeres.