Loss of Ku’s DNA end binding activity affects telomere length via destabilizing telomere-bound Est1 rather than altering TLC1 homeostasis

Saccharomyces cerevisiae telomerase, which maintains telomere length, is comprised of an RNA component, TLC1, the reverse transcriptase, Est2, and regulatory subunits, including Est1. The Yku70/Yku80 (Ku) heterodimer, a DNA end binding (DEB) protein, also contributes to telomere length maintenance. Ku binds TLC1 and telomere ends in a mutually exclusive fashion, and is required to maintain levels and nuclear localization of TLC1. Ku also interacts with Sir4, which localizes to telomeres. Here we sought to determine the role of Ku’s DEB activity in telomere length maintenance by utilizing yku70-R456E mutant strains, in which Ku has reduced DEB and telomere association but proficiency in TLC1 and Sir4 binding, and TLC1 nuclear retention. Telomere lengths in a yku70-R456E strain were nearly as short as those in yku∆ strains and shorter than in strains lacking either Sir4, Ku:Sir4 interaction, or Ku:TLC1 interaction. TLC1 levels were decreased in the yku70-R456E mutant, yet overexpression of TLC1 failed to restore telomere length. Reduced DEB activity did not impact Est1’s ability to associate with telomerase but did result in decreased association of Est1 with the telomere. These findings suggest Ku’s DEB activity maintains telomere length homeostasis by preserving Est1’s interaction at the telomere rather than altering TLC1 levels.