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 Ku9s telomere association in different populations of cen3tel cells, which had a wide range of average telomere lengths, supported Ku9s localization at human telomeres primarily via protein-protein interaction. We also found that the Ku70 and Ku80 α5 helices, which are on opposing sides of the heterodimer and were previously implicated in S. cerevisiae Ku9s NHEJ and telomeric functions, respectively, participated in Ku9s telomere association in human cells. While the Ku70 α5 mutant showed increased interaction with TRF2, the Ku80 α5 mutant was not impacted for TRF2 association. Interestingly, residues altered to impair Ku9s 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.