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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.

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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.
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fig6: 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.

Mentions: To explore the role that Ku’s interaction with Est1 may play in telomere length maintenance, we examined the impact of Ku on Est1’s association with telomeres. It was previously shown that both Est1 and Est2 telomere association is reduced in the absence of Ku–TLC1 interaction (Fisher et al. 2004; Chan et al. 2008). However, robust association of Est1 and Est2 are mutually dependent, with the absence of one subunit resulting in reduced telomere association of the other (Chan et al. 2008). Therefore, it is difficult to determine whether Ku is important for recruitment of one or both of the subunits. As telomere association of Est2 in G1 is completely dependent on Ku (Fisher et al. 2004), one hypothesis is that Ku is required for efficient Est2 recruitment and the reduction of Est1 at telomeres is a secondary effect of less Est2 present. The results with the Cdc13 fusion proteins, however, do not support this hypothesis as tethering Est2 to telomeres in the absence of Ku–TLC1 interaction or Ku could not bypass the role of Ku in promoting telomere elongation (Figure 1C). Furthermore, tethering Est1 to telomeres is sufficient for extensive telomere elongation in the absence of Ku–TLC1 interaction or Ku (Figure 3A), suggesting that a major role of Ku may be to promote Est1 recruitment to telomeres. To determine if Ku influences Est1’s telomere association independently of promoting Est2 recruitment, ChIP experiments were performed in WT, yku80∆, and yku80-135i strains expressing a Cdc13–Est2 fusion. To facilitate immunoprecipitation, endogenous Est1 was C-terminally tagged with a 13×-myc epitope. We found that both yku80∆ and yku80-135i mutants had reduced telomere association of Est1 compared to WT strains when a Cdc13–Est2 fusion was expressed (Figure 6, A and B). The decrease in Est1 telomere association could not be explained by differences in Est1 protein level or immunoprecipitation efficiency in Ku mutant strains as Est1 protein levels were equivalent in input and IP samples (Figure 6C).


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)

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.
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fig6: 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.
Mentions: To explore the role that Ku’s interaction with Est1 may play in telomere length maintenance, we examined the impact of Ku on Est1’s association with telomeres. It was previously shown that both Est1 and Est2 telomere association is reduced in the absence of Ku–TLC1 interaction (Fisher et al. 2004; Chan et al. 2008). However, robust association of Est1 and Est2 are mutually dependent, with the absence of one subunit resulting in reduced telomere association of the other (Chan et al. 2008). Therefore, it is difficult to determine whether Ku is important for recruitment of one or both of the subunits. As telomere association of Est2 in G1 is completely dependent on Ku (Fisher et al. 2004), one hypothesis is that Ku is required for efficient Est2 recruitment and the reduction of Est1 at telomeres is a secondary effect of less Est2 present. The results with the Cdc13 fusion proteins, however, do not support this hypothesis as tethering Est2 to telomeres in the absence of Ku–TLC1 interaction or Ku could not bypass the role of Ku in promoting telomere elongation (Figure 1C). Furthermore, tethering Est1 to telomeres is sufficient for extensive telomere elongation in the absence of Ku–TLC1 interaction or Ku (Figure 3A), suggesting that a major role of Ku may be to promote Est1 recruitment to telomeres. To determine if Ku influences Est1’s telomere association independently of promoting Est2 recruitment, ChIP experiments were performed in WT, yku80∆, and yku80-135i strains expressing a Cdc13–Est2 fusion. To facilitate immunoprecipitation, endogenous Est1 was C-terminally tagged with a 13×-myc epitope. We found that both yku80∆ and yku80-135i mutants had reduced telomere association of Est1 compared to WT strains when a Cdc13–Est2 fusion was expressed (Figure 6, A and B). The decrease in Est1 telomere association could not be explained by differences in Est1 protein level or immunoprecipitation efficiency in Ku mutant strains as Est1 protein levels were equivalent in input and IP samples (Figure 6C).

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