Analysis of the Role of EST1 in Yeast Telomerase

摘要

I have taken two complementary approaches towards characterizing the role of the Est1 telomerase subunit in S. cerevisiae telomere replication. The first method utilized a unique set of Cdc13-telomerase fusion proteins; this study led to the proposal that Est1 and Cdc13 (a telomere end-binding protein) are co-mediators of telomerase access. The role for Est1 in telomerase recruitment is strongly supported by the demonstration that telomeres can be stably maintained in the absence of Est1, by fusing Cdc13 directly to the catalytic subunit of telomerase, Est2. These studies further suggest that Est1 has a second role in telomere replication (based on the observation that the Cdc13-Est2 fusion was unable to promote telomere elongation in the absence of Est1) . To explore this second role further, I isolated a set of alanine-scan mutations in Est1 that retained association with telomerase but displayed telomere replication defects, and identified an est1 mutant that displayed phenotypes that were distinct from recruitment defective est1 alleles. The identification of two distinct classes of mutants, both of which associate with an active telomerase enzyme but which appear to perturb different aspects of Est1 function, supports the idea that Est1 has two roles in telomere replication.