Cdc13 Cooperates with the Yeast Ku Proteins and Stn1 To Regulate Telomerase Recruitment

Abstract

The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both onSTN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.

ACKNOWLEDGMENTS

We thank James Haber, Sang Eun Lee, Victoria Lundblad, Thomas Petes, Patricia Greenwell, Ethelle Moustacchi, Daniel Gottschling, Miriam Singer, Leland Hartwell, and Eric Gilson for the gifts of strains and plasmids. We also thank Eric Gilson for discussion, Catherine Koering for technical advice concerning the band shift experiments, and Suzy Markossian and Armelle Roisin for operating the semiautomated DNA sequencer.

This work was supported by grants from the Association pour la Recherche contre le Cancer, the Centre National de la Recherche Scientifique, programme Génome, the Comités Départementaux de l'Ardèche, la Loire et la Haute-Savoie de la Ligue Nationale contre le Cancer, and the Région Rhône-Alpes, programme Apoptose et Vieillissement.