This chapter focuses on the characterization of the Saccharomyces cerevisiae
Pif1p helicase. Pif1p is the prototype member of the PIF1 family of helicases, which is conserved from yeast to human (1
). Two isoforms of the enzyme are expressed in yeast, owing to alternative usage of two start codons from the same mRNA. Translation from the first start codon leads to the synthesis of a mitochondria-directed isoform, while translation from the second AUG codon leads to the synthesis of the nuclear isoform (3
). Genetic studies have shown that the nuclear form of Pif1p plays an important role in counteracting the activity of telomerase, the specialized reverse transcriptase that elongates the end of eukaryotic chromosomes. Through this activity, Pif1p prevents gross chromosomal rearrangements that are due to the addition of telomerase-mediated de novo telomere addition at double strand breaks (4
). In vivo and in vitro data suggest that this action is achieved through a direct interaction between Pif1p and telomerase (5
). Using oligonucleotide-based radiometric assays, Pif1p has been shown to unwind preferentially RNA–DNA hybrids over DNA substrates (7
). This preference suggests that Pif1p inhibits telomerase by unwinding the RNA–DNA substrate formed by the telomerase RNA, TLC1, and the telomeric DNA end. Importantly, the interaction between Pif1p and telomerase is conserved in evolution, since Pif1p has been shown to interact with mouse and human telomerase (8–
10). This chapter focuses on in vitro methods to purify recombinant yeast Pif1p and to characterize its activity by classical oligonucletide substrate-based radiometric assays. We also describe methods to study in vitro its functional interaction with yeast telomerase.