The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres

Abstract

Telomere length is tightly regulated in cells that express telomerase. The Saccharomyces cerevisiae Ku heterodimer, a DNA end-binding complex, positively regulates telomere length in a telomerase-dependent manner. Ku associates with the telomerase RNA subunit TLC1, and this association is required for TLC1 nuclear retention. Ku–TLC1 interaction also impacts the cell-cycle-regulated association of the telomerase catalytic subunit Est2 to telomeres. The promotion of TLC1 nuclear localization and Est2 recruitment have been proposed to be the principal role of Ku in telomere length maintenance, but neither model has been directly tested. Here we study the impact of forced recruitment of Est2 to telomeres on telomere length in the absence of Ku’s ability to bind TLC1 or DNA ends. We show that tethering Est2 to telomeres does not promote efficient telomere elongation in the absence of Ku–TLC1 interaction or DNA end binding. Moreover, restoration of TLC1 nuclear localization, even when combined with Est2 recruitment, does not bypass the role of Ku. In contrast, forced recruitment of Est1, which has roles in telomerase recruitment and activation, to telomeres promotes efficient and progressive telomere elongation in the absence of Ku–TLC1 interaction, Ku DNA end binding, or Ku altogether. Ku associates with Est1 and Est2 in a TLC1-dependent manner and enhances Est1 recruitment to telomeres independently of Est2. Together, our results unexpectedly demonstrate that the principal role of Ku in telomere length maintenance is to promote the association of Est1 with telomeres, which may in turn allow for efficient recruitment and activation of the telomerase holoenzyme.

Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process

“…1b). Many proteins including the Ku complex (composed of Ku70 and Ku80) and telomerase contribute to a perinuclear telomere-tethering process that is somewhat distinct from cohibin-dependent tethering 13,20,37,38 . Since Ku disruption is ineffectual in our subtelomeric DSB survival assay (Fig.…”

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

“…In addition, we found the telomeres in the yku70-R456E mutant were also shorter than in tlc1∆-48 (as previously reported 44 ) and yku80-135i strains. While the shorter telomere lengths in yku80-135i and tlc1∆-48 mutants as compared to sir4∆, yku80-L111R, and yku80-L115A mutants has been explained by the additional loss of TLC1 nuclear retention 14 , this could not explain the even shorter telomeres in the yku70-R456E mutant, as TLC1 is still retained the nucleus in this mutant strain 33 .…”

“…Because the presence of Est1 at the telomere decreases in the absence of Ku ( Fig. 6 and ref 33 ), it is likely that the increase in Est1:Cdc13 interaction observed represented complexes not associated with the telomere end. Additionally, the yku80-135i mutant, which maintains binding to the DNA end 31 , had levels of soluble Est:Cdc13 complexes similar to wild type ( Fig.…”

“…One model involves Ku's impact on the subcellular localization and steady state levels of TLC1. Ku associates with a specific stem-loop structure on TLC1 30,31 ; this binding is required for the nuclear retention of TLC1 as cells lacking Ku or Ku:TLC1 interaction (such as yku80-135i or tlc1∆-48 mutants) fail to retain TLC1 in the nucleus 32,33 . Additionally, steady state levels of TLC1 are decreased in yku∆, yku80-135i, and tlc1∆-48 mutants, although the mechanism responsible for this reduction remains unknown 34,35 .…”

“…Nonetheless, Ku's DEB activity is required for its influence on telomere length 44 . The yku70-R456E mutant, which has reduced binding to DNA and the telomere end, has stably short telomeres, despite binding TLC1 and retaining its localization in the nucleus like wild type strains 33,44 . Additionally, the yku70-R456E mutant is proficient for Sir4 7 binding suggesting that loss of Ku's DEB activity alone is sufficient to cause the short telomere phenotype 44 .…”