The principal role of Ku in telomere length maintenance is promotion of Est1 association with telomeres.
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.
Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.Show MeSH
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Mentions: After finding that Ku was unable to promote extensive telomere elongation in the presence of a Cdc13–Est2 fusion in the absence of TLC1 binding, we wanted to know if Ku’s interaction with TLC1 in the absence of DNA binding was sufficient to promote telomere elongation. The ability of Ku–TLC1 interaction to promote telomere elongation in the absence of DNA binding was plausible because Ku cannot bind TLC1 and DNA ends simultaneously (Pfingsten et al. 2012). To test the requirement of DNA binding to promote extensive telomere elongation in the presence of a Cdc13–Est2 fusion, we made use of a previously characterized DNA-binding-defective allele of Yku70, yku70-R456E (Lopez et al. 2011). The single amino acid substitution in the DNA-binding channel of Yku70 results in a Ku heterodimer that can no longer efficiently bind DNA ends but still associates with TLC1in vivo as determined by co-immunoprecipitation (Lopez et al. 2011). However, much like the yku80-135i and yku80∆ strains, telomeres did not extensively elongate when a Cdc13–Est2 fusion was expressed in the absence of DNA binding by Ku (Figure 2A, yku70-R456E strain). The reduced telomere elongation was unlikely due to TLC1 mislocalization in the cytoplasm as TLC1 was mainly nuclear in a yku70-R456E strain (Figure 2B). These data suggest that, although Ku does not associate with RNA and DNA simultaneously, its role in telomere length maintenance requires it to retain the ability to bind both TLC1 and DNA ends.
Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.