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The yeast Pif1p helicase removes telomerase from telomeric DNA

Nature volume 438, pages 57–61 (2005)Cite this article

Abstract

Telomeres are the physical ends of eukaryotic chromosomes. Genetic studies have established that the baker's yeast Pif1p DNA helicase is a negative regulator of telomerase, the specialized reverse transcriptase that maintains telomeric DNA, but the biochemical basis for this inhibition was unknown. Here we show that in vitro, Pif1p reduces the processivity of telomerase and releases telomerase from telomeric oligonucleotides. The released telomerase is enzymatically active because it is able to lengthen a challenger oligonucleotide. In vivo, overexpression of Pif1p reduces telomerase association with telomeres, whereas depleting cells of Pif1p increases the levels of telomere-bound Est1p, a telomerase subunit that is present on the telomere when telomerase is active. We propose that Pif1p helicase activity limits telomerase action both in vivo and in vitro by displacing active telomerase from DNA ends.

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Figure 1: Activity of Pif1p.
Figure 2: Pif1p reduces telomerase processivity and promotes lengthening of challenger primers.
Figure 3: Pif1p overexpression decreases Est2p and Est1p telomere association.
Figure 4: Pif1p depletion increases telomere association of Est1p.

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Acknowledgements

We thank A. Chan for help with some of the experiments and J. Cooper, T. Fisher and M. Sabourin for comments on the manuscript. This work was supported by the NIH. J.B.B. was supported in part by a fellowship from the Association de la Recherche contre le Cancer and in part from a fellowship from the NJ Commission on Cancer Research; L.R.V. was supported in part by a Helen Hay Whitney post doctoral fellowship and in part by an NIH grant. Author Contributions J.-B.B. and L.R.V. contributed equally to this work.

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Author notes

  1. Leticia R. Vega

    Present address: Barry University, School of Natural and Health Sciences, Miami Shores, Florida, 33161, USA

  2. Jean-Baptiste Boulé and Leticia R. Vega: *These authors contributed equally to this work

Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, New Jersey, 08544, Princeton, USA

    Jean-Baptiste Boulé, Leticia R. Vega & Virginia A. Zakian

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  1. Jean-Baptiste Boulé
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  2. Leticia R. Vega
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  3. Virginia A. Zakian
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Correspondence to Virginia A. Zakian.

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Supplementary information

Supplementary Methods

Detailed protocol of Pif1p overexpression in bacteria and Pif1p purification. Detailed protocol of the preparation of telomerase extracts. (DOC 26 kb)

Supplementary Figure S1

Schematic drawing describing how Pif1p uses its ATPase activity to inhibit telomerase. (PDF 145 kb)

Supplementary Figure S2

Binding of Pif1p and Pif1p-K264A to single stranded DNA as assessed by bandshift experiments. (PDF 99 kb)

Supplementary Figure S3

Inhibition of telomerase activity at various Pif1p concentrations in vitro. (PDF 308 kb)

Supplementary Figure S4

In vitro release by Pif1p of Est2p bound to immobilized telomeric oligonucleotides. (PDF 129 kb)

Supplementary Figure S5

Lack of effect of Pif1p depletion on the association of Est2p or Cdc13p on telomere ends in vivo. (PDF 150 kb)

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Boulé, JB., Vega, L. & Zakian, V. The yeast Pif1p helicase removes telomerase from telomeric DNA. Nature 438, 57–61 (2005). https://doi.org/10.1038/nature04091

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