Loss of Ku\u2019s DNA end binding activity affects telomere length via destabilizing telomere-bound Est1 rather than altering TLC1 homeostasis

Laramie D Lemon,Alison A Bertuch,Danna K Morris

doi:10.1038/s41598-019-46840-2

Laramie D Lemon, Alison A Bertuch + Show 1 more

Open Access

https://doi.org/10.1038/s41598-019-46840-2

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Journal: Scientific Reports	Publication Date: Jul 23, 2019
Citations: 4	License type: open-access

Affiliation: Baylor College of Medicine

Abstract

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.

Highlights

Telomeres are nucleoprotein complexes that cap the natural ends of linear eukaryotic chromosomes[1]
We found the telomeres in the yku70-R456E mutant were much shorter than in the sir4∆, yku80-L111R, and yku80-L115A strains
While the shorter telomere lengths in yku80-135i and tlc1∆-48 mutants as compared to sir4∆, yku80L111R, 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]

Summary

Introduction

Telomeres are nucleoprotein complexes that cap the natural ends of linear eukaryotic chromosomes[1]. The length of telomeric DNA, including sequence lost due to the end-replication problem, can be maintained by the enzyme telomerase, a specialized reverse transcriptase that has an integral RNA component that provides the template for de novo synthesis of telomere repeats onto native chromosome ends[5]. Est[1] is involved in the recruitment of telomerase to the telomere during late S phase through its association with Cdc[13], a single-stranded DNA binding protein that binds the terminal 3′ overhang of the telomeric G-rich strand with high affinity[14,15,16,17,18,19]. The role of Est[3] in telomere maintenance remains unclear, but it has been hypothesized that part of Est1’s activation function involves the recruitment of Est[3] to the telomere[21]. In contrast to strains lacking telomerase components, Ku deficient strains have stably short telomeres and do not senesce

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