Characterization of the helicase activity and anti-telomerase properties of yeast Pif1p in vitro - PubMed (original) (raw)

Characterization of the helicase activity and anti-telomerase properties of yeast Pif1p in vitro

Jean-Baptiste Boulé et al. Methods Mol Biol. 2010.

Abstract

Pif1p is the prototype member of a family of helicases that is highly conserved from yeast to humans. In yeast, Pif1p is involved in many aspects of the preservation of genome stability. In particular, Pif1p is involved in the maintenance of mitochondrial DNA and in the direct inhibition of telomerase at telomeres and double-stranded breaks. Here we describe methods to purify Pif1p and study in vitro its enzymatic properties and functional interaction with telomerase.

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Figures

Fig. 25.1

Pif1p purification. Coomassie staining of an 8% SDS-PAGE gel showing the supernatant after IPTG induction (SN), the protein pool after elution from the Talon resin (His), or the protein pool after cation exchange (CE) chromatography.

Fig. 25.2

Effects of Pif1p on telomerase activity in vitro. Autoradiograph of a sequencing gel showing telomerase extension products of a TEL15 oligonucleotide in the presence of telomerase alone (lane 1), in presence of RNase A (lane 2), or two concentrations of Pif1p (lane 3, 1 μM Pif1p; lane 4, 0.2 μM Pif1p). LC: loading control.

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