Limits...
The principal role of Ku in telomere length maintenance is promotion of Est1 association with telomeres.

Williams JM, Ouenzar F, Lemon LD, Chartrand P, Bertuch AA - Genetics (2014)

Bottom Line: 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.Moreover, restoration of TLC1 nuclear localization, even when combined with Est2 recruitment, does not bypass the role of Ku.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.

Show MeSH

Related in: MedlinePlus

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).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4125388&req=5

fig5: 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).

Mentions: The ability of the Cdc13–Est1 fusion to bypass the role of Ku suggests that Ku’s primary contribution to telomere length maintenance is via Est1. In further support of an interaction between Ku and Est1, we found that myc-tagged Est1 co-immunoprecipitated with flag-tagged Yku80 in asynchronous cells (Figure 5A). This interaction was dependent on the ability of Ku to bind TLC1 as the Ku–Est1 interaction was reduced when a yku80-135i allele was expressed (Figure 5A). Using a strain harboring identically myc-tagged Est1 and Est2, we found that Est2, in addition to Est1, co-immunoprecipitated with flag-tagged Yku80 (Figure 5, B–D). Consistent with the interaction between Ku and Est1 being dependent upon Ku’s ability to bind TLC1, treatment of lysates with RNase A abolished the co-immunoprecipitation of Est1 and Est2 with Yku80 (Figure 5B). Furthermore, the interaction between Ku and Est1 and Est2 was not mediated by DNA as treatment of lysates with DNase I had no impact on the co-immunoprecipitation efficiency (Figure 5C and Figure S3A). Although no genomic DNA was detected in the lysates used in this experiment, samples were treated under conditions sufficient to degrade 3 μg of a 1.2-kb PCR fragment (Figure S3B). Therefore, any endogenous DNA in the extracts should have been degraded.


The principal role of Ku in telomere length maintenance is promotion of Est1 association with telomeres.

Williams JM, Ouenzar F, Lemon LD, Chartrand P, Bertuch AA - Genetics (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125388&req=5

fig5: 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).
Mentions: The ability of the Cdc13–Est1 fusion to bypass the role of Ku suggests that Ku’s primary contribution to telomere length maintenance is via Est1. In further support of an interaction between Ku and Est1, we found that myc-tagged Est1 co-immunoprecipitated with flag-tagged Yku80 in asynchronous cells (Figure 5A). This interaction was dependent on the ability of Ku to bind TLC1 as the Ku–Est1 interaction was reduced when a yku80-135i allele was expressed (Figure 5A). Using a strain harboring identically myc-tagged Est1 and Est2, we found that Est2, in addition to Est1, co-immunoprecipitated with flag-tagged Yku80 (Figure 5, B–D). Consistent with the interaction between Ku and Est1 being dependent upon Ku’s ability to bind TLC1, treatment of lysates with RNase A abolished the co-immunoprecipitation of Est1 and Est2 with Yku80 (Figure 5B). Furthermore, the interaction between Ku and Est1 and Est2 was not mediated by DNA as treatment of lysates with DNase I had no impact on the co-immunoprecipitation efficiency (Figure 5C and Figure S3A). Although no genomic DNA was detected in the lysates used in this experiment, samples were treated under conditions sufficient to degrade 3 μg of a 1.2-kb PCR fragment (Figure S3B). Therefore, any endogenous DNA in the extracts should have been degraded.

Bottom Line: 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.Moreover, restoration of TLC1 nuclear localization, even when combined with Est2 recruitment, does not bypass the role of Ku.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.

Show MeSH
Related in: MedlinePlus