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
Related in: MedlinePlus
Mentions: It has been previously demonstrated that, in an est1∆ strain, expression of a Cdc13–Est2 fusion can bypass Est1’s telomerase recruitment function, resulting in approximately WT length telomeres (Evans and Lundblad 1999). Furthermore, the Cdc13–Est2 fusion was unable to promote extensive, progressive elongation in an est1∆ strain, leading to the conclusion that Est1 has a telomerase activation function (Evans and Lundblad 1999). The inability of the Cdc13–Est2 fusion to promote telomere elongation to the same extent as in a WT strain in yku80-135i or yku80∆ mutants suggests that Ku may also have a role in telomerase activation. Additionally, the ability of the Cdc13–Est1 fusion to promote progressive telomere elongation in the absence of Ku indicates that Ku’s role in telomere length maintenance may be primarily through an Est1-dependent pathway. To test this, we performed epistasis analysis of EST1 and the yku80-135i mutation in the presence of a Cdc13–Est2 fusion. To enable recovery of all genotypes of interest from a single parental stain, EST1/est1∆ yku80∆/yku80-135i CDC13/cdc13∆ diploid strains harboring both YKU80 and CDC13-EST2 plasmids were sporulated and dissected, ensuring similar initial telomere lengths. After dissection of 20 tetrads, however, no est1∆ yku80∆ double mutants were recovered even in the presence of a Cdc13–Est2 fusion. This was presumably due to the previously reported synthetic lethality of est1∆ yku80∆ mutations (Nugent et al. 1998) and increased single-stranded DNA that cannot be rescued by a Cdc13–Est2 fusion (Tong et al. 2011). Telomere length analysis of recovered haploid genotypes revealed, as expected, extensive telomere elongation in the WT strain expressing a Cdc13–Est2 fusion whereas telomeres were stably maintained but not elongated in the absence of Est1 (Figure 7). Interestingly, combining est1∆ and yku80-135i mutations had a slightly additive negative effect, with telomeres shorter in the est1∆ yku80-135i strain than in the est1∆ strain in the presence of a Cdc13–Est2 fusion (Figure 7). Although tethering Est1 to telomeres is sufficient to bypass the role of Ku in promoting telomere elongation, the est1∆ yku80-135i epistasis results demonstrate a specific contribution of Ku–TLC1 interaction that is independent of Est1. However, deletion of Est1 has a much greater impact on telomere lengthening in the presence of a Cdc13–Est2 fusion than deletion of YKU80 (Figure 7, est1∆ strain compared to yku80∆ strain), suggesting that any contribution that Ku may have in telomerase activation is minor compared to that of Est1.
Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.