In Saccharomyces cerevisiae, a Cdc13-Est1 interaction is proposed to mediate recruitment of telomerase to DNA ends. Here we provide unique in vitro evidence for this model by demonstrating a direct interaction between purified Cdc13 and Est1. The Cdc13-Est1 interaction is specific and requires the in vivo defined Cdc13 recruitment domain. Moreover, in the absence of this interaction, Est1 is excluded from telomeric single-stranded (ss)DNA. The apparent association constand (Kd) between Est1 and a Cdc13-telomeric ssDNA complex was ∼250 nM. In G2 phase cells, where telomerase is active, Cdc13 and Est1 were sufficiently abundant (∼420 and ∼110 copies per cell, respectively) to support complex formation. Interaction between Cdc13 and Est1 was unchanged by three telomerase-deficient mutations, Cdc13E252K (cdc13-2), Est1K444E (est1-60), and Cdc13S249,255D, indicating that their telomerase null phenotypes are not due to loss of the Cdc13-Est1 interaction. These data recapitulate in vitro the first step in telomerase recruitment to telomeric ssDNA and suggest that this step is necessary to recruit telomerase to DNA ends.
|Original language||English (US)|
|Number of pages||8|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Dec 20 2011|
All Science Journal Classification (ASJC) codes