title: The telomeric Cdc13 protein interacts directly with the telomerase subunit Est1 to bring it to telomeric DNA ends in vitro.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wu
authors.forename: Virginia A
authors.initials: VA
authors.lastname: Zakian
mesh_terms: D000818:Animals; D002453:Cell Cycle; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D016615:Telomere / Q000648:ultrastructure*; D034501:Telomere-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C452219:Cdc13 protein, S cerevisiae; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins; D004247:DNA; C099024:EST1 protein, S cerevisiae; D019098:Telomerase