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RPA regulates telomerase action by providing Est1p access to chromosome ends

Nature Genetics volume 36, pages 46–54 (2004)Cite this article

Abstract

Replication protein A (RPA) is a highly conserved single-stranded DNA–binding protein involved in DNA replication, recombination and repair. We show here that RPA is present at the telomeres of the budding yeast Saccharomyces cerevisiae, with a maximal association in S phase. A truncation of the N-terminal region of Rfa2p (associated with the rfa2Δ40 mutated allele) results in severe telomere shortening caused by a defect in the in vivo regulation of telomerase activity. Cells carrying rfa2Δ40 show impaired binding of the protein Est1p, which is required for telomerase action. In addition, normal telomere length can be restored by expressing a Cdc13-Est1p hybrid protein. These findings indicate that RPA activates telomerase by loading Est1p onto telomeres during S phase. We propose a model of in vivo telomerase action that involves synergistic action of RPA and Cdc13p at the G-rich 3′ overhang of telomeric DNA.

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Figure 1: Rfa2p binds to telomeric DNA in vivo.
Figure 2: Rfa2p is involved in telomere length regulation.
Figure 3: RFA2 acts in the telomerase pathway.
Figure 4: Telomeric association of Rfa2p (or Rfa2Δ40p) and Rfa1p in wild-type (WT) and rfa2Δ40 cells.
Figure 5: The rfa2Δ40 allele impairs Est1p telomeric DNA binding during S phase.
Figure 6: Association of Est1-myc, Est2-myc and Cdc13-myc in yKu70Δ cells.
Figure 7: Telomere lengthening by targeted Est1p in rfa2Δ40 cells.
Figure 8: Model of telomerase action during the cell cycle.

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Acknowledgements

We thank S. Brill for antibodies to Rfa2p, V. Lundblad for providing plasmid pVL1120 and plasmids used to Myc-tag Cdc13p and Est2p, and V. Zakian for reagents for the one-hybrid assay for telomeric proteins. Work in V.G.'s laboratory was supported by l'Association pour la Recherche sur le Cancer and by la Fondation pour la Recherche, work in E.G.'s laboratory by the Ligue Nationale contre le Cancer and work in M.P.L.'s laboratory by the Associazione Italiana per la Ricerca sul Cancro and by Telethon-Italy.

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  1. Vera Schramke and Pierre Luciano: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Centre Nationale de la Recherche Scientifique (CNRS), 31 chemin Joseph Aiguier, Marseille, 13402 Cedex 20, France

    Vera Schramke, Pierre Luciano, Sylvine Guillot, Yves Corda & Vincent Géli

  2. Laboratoire de Biologie Moléculaire de la Cellule, CNRS, Unité Mixte de Recherche 5161, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, Lyon, 69364, Cedex, France

    Vanessa Brevet & Eric Gilson

  3. Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, Milano, 20126, Italy

    Maria Pia Longhese

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Correspondence to Vincent Géli.

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Schramke, V., Luciano, P., Brevet, V. et al. RPA regulates telomerase action by providing Est1p access to chromosome ends. Nat Genet 36, 46–54 (2004). https://doi.org/10.1038/ng1284

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