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.

Keywords: Est1; Est2; Ku; TLC1; telomerase; telomere.

Figure 1

Telomerase overexpression or Cdc13-Est2 expression has differential effects in WT, yku80-135i, and yku80∆ strains. (A) Telomere length analysis by Southern blot of XhoI-digested DNA isolated from WT, yku80-135i, and yku80∆ strains transformed with Est1 and Est2 overexpression plasmids, singly and in combination. (B) Telomere length analysis by Southern blotting of 1×–4× serial single-colony streakouts of WT, yku80-135i, and yku80∆ strains simultaneously overexpressing Est1, Est2, and TLC1. (C) Telomere length analysis of 1×–5× serial single-colony streakouts of cdc13∆ (WT), cdc13∆ yku80-135i (yku80-135i), and cdc13∆ yku80∆ (yku80∆) strains expressing a Cdc13–Est2 fusion.

Figure 2

TLC1 nuclear retention in addition to Est2 recruitment cannot bypass the role of Ku. (A) Telomere length analysis of 2×–6× serial single-colony streakouts of cdc13∆ (WT), cdc13∆ yku70-R456E (yku70-R456E), and cdc13∆ yku70∆ (yku70∆) strains expressing a Cdc13–Est2 fusion. (B) Quantification of TLC1 localization by FISH in a yku70-R456E strain. Error bars represent ± 1 SD. Unbudded cells from asynchronous cultures were analyzed. However, TLC1 localization was similar in all cells (data not shown). (C) Telomere length analysis of 2×–6× serial single-colony streakouts of cdc13∆ (WT), cdc13∆ yku70∆ (yku70∆), and cdc13∆ yku70∆ exo1∆ (yku70∆ exo1∆) strains expressing a Cdc13–Est2 fusion.

Figure 3

Tethering Est1 to telomeres promotes efficient telomere elongation in Ku mutant strains. (A) Telomere length analysis by Southern blot of XhoI-digested DNA isolated from 1×–5× serial single-colony streakouts of cdc13∆ (WT), cdc13∆ yku80-135i (yku80-135i), and cdc13∆ yku80∆ (yku80∆) strains expressing a Cdc13–Est1 fusion. (B) Telomere length analysis of 2×–6× serial single-colony streakouts of cdc13∆ (WT), cdc13∆ yku70-R456E (yku70-R456E), and cdc13∆ yku70∆ (yku70∆) strains expressing a Cdc13–Est1 fusion.

Figure 4

Cdc13-Est2 or Cdc13-Est1 expression partially rescues TLC1 nuclear localization in yku80-135i and yku80∆ strains. (A) Quantification of TLC1 localization by FISH in cdc13∆ (WT), cdc13∆ yku80-135i (yku80-135i), and cdc13∆ yku80∆ strains expressing either a Cdc13–Est2 or Cdc13–Est1 fusion protein. Error bars represent ± 1 SD. Unbudded cells from asynchronous cultures were analyzed. However, TLC1 localization was similar in all cells (data not shown). (B) Telomere length analysis of 1×–3× serial single-colony streakouts of cdc13∆ (WT), cdc13∆ yku80-135i (yku80-135i), and cdc13∆ yku80∆ (yku80∆) strains expressing a Cdc13–Est2 or Cdc13–Est1 fusion.

Figure 5

Ku associates with Est1 and Est2 in a TLC1-dependent manner. (A) Co-immunoprecipitation of Est1-myc with Yku80-FLAG and Yku80-135i-FLAG. Anti-FLAG immunoprecipitations were performed with whole-cell extracts of indicated strains. Inputs and IPs were analyzed by Western blotting with α-myc to detect Est1 and with α-FLAG to detect Yku80 or Yku80-135i. Inputs were also probed with α-PGK as a loading control. (B) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in RNase A-treated and untreated extracts. Western blots were probed with α-myc to detect Est1 (bottom band) and Est2 (top band). (C) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in DNase I-treated and untreated extracts. (D) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in asynchronous, α-factor-, hydroxyurea-, and nocodazole-arrested cells. Quantification of relative amount of Est1 in inputs and immunoprecipitates represents the average and standard deviation of four independent experiments. (E) Co-immunoprecipitation of Est1 with Est2 (EST1-MYC FLAG-MYC-EST2 strain) or Yku80 (EST1-MYC MYC-EST2 YKU80-FLAG strain).

Figure 6

The association of Est1 with telomeres is dependent on Ku–TLC1 interaction even when Est2 is tethered to the telomere. (A) Myc-tagged Est1 was immunoprecipitated from formaldehyde cross-linked cdc13∆ (WT), cdc13∆ yku80∆ (yku80∆), and cdc13∆ yku80-135i (yku80-135i) strains expressing a Cdc13–Est2 fusion. Isolated DNA was dot-blotted onto a membrane and probed with a radiolabeled TyB (inputs) or telomere-specific T-G_(1-3) (IPs) probe. (B) Graphical representation of the average IP/input signal relative to the no-tag control strain based on four independent experiments. Error bars represent ± 1 SD. (C) Western blot showing equivalent amounts of Est1 protein immunoprecipitated from cdc13∆ (WT), cdc13∆ yku80∆ (yku80∆), and cdc13∆ yku80-135i (yku80-135i) strains expressing a Cdc13–Est2 fusion. A total of 100 μg of whole-cell extract prior to immunoprecipitation (input) was loaded, demonstrating that Est1 protein level is equal in all three strains.

Figure 7

Ku has a minor role in telomere length maintenance that is independent of Est1. Telomere length analysis by Southern blot of 1×–3× serial single-colony streakouts of haploid strains cdc13∆ (WT), cdc13∆ est1∆ (est1∆), cdc13∆ est1∆ yku80-135i (est1∆ yku80-135i), and cdc13∆ yku80∆ (yku80∆) strains expressing a Cdc13–Est2 fusion dissected from a EST1/est1∆ yku80∆/yku80-135i CDC13/cdc13∆ diploid strain harboring both YKU80 and CDC13-EST2 plasmids.