Telomerase is a telomere dedicated reverse transcriptase that replicates the very ends of eukaryotic chromosomes. Saccharomyces cerevisiae telomerase consists of TLC1 (the RNA template), Est2 (the catalytic subunit), and two accessory proteins, Est1 and Est3, that are essential in vivo for telomerase activity but are dispensable for catalysis in vitro. Est1 functions in both recruitment and activation of telomerase. The association of Est3 with telomeres occurred largely in late S/G2 phase, the time when telomerase acts and Est1 telomere binding occurs. Est3 telomere binding was Est1-dependent. This dependence is likely due to a direct interaction between the two proteins, as purified recombinant Est1 and Est3 interacted in vitro. Est3 abundance was neither cell cycle–regulated nor Est1-dependent. Est3 was the most abundant of the three Est proteins (84.3±13.3 molecules per cell versus 71.1±19.2 for Est1 and 37.2±6.5 for Est2), so its telomere association and/or activity is unlikely to be limited by its relative abundance. Est2 and Est1 telomere binding was unaffected by the absence of Est3. Taken together, these data indicate that Est3 acts downstream of both Est2 and Est1 and that the putative activation function of Est1 can be explained by its role in recruiting Est3 to telomeres.
Owing to the biochemical properties of DNA polymerases, the free ends of linear chromosomes, called telomeres, cannot be replicated by the same mechanisms that suffice for the rest of the chromosome. Instead they are maintained by a telomere-dedicated reverse transcriptase called telomerase that uses its integral RNA component as the template to make more telomeric DNA. In baker's yeast, telomerase is composed of a catalytic subunit (Est2), the templating RNA (TLC1), and two accessory proteins, Est1 and Est3. Here we show that Est3 associates with telomeres late in the cell cycle, at the same time when telomerase is active, and this binding was Est1-dependent, even though Est3 abundance was neither cell cycle–regulated nor Est1-dependent. Since purified Est3 and Est1interacted in vitro, Est1-dependent recruitment of Est3 is probably due to direct protein–protein interaction. Neither Est1 nor Est2 telomere binding was Est3-dependent. Thus, Est3 acts downstream of telomerase recruitment to promote telomerase activity, and the telomerase activation functions of Est1 can be explained by its recruiting Est3 to telomeres.