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 (K(d)) 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, Cdc13(E252K) (cdc13-2), Est1(K444E) (est1-60), and Cdc13(S249,255D), 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.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|