Characterization of the helicase activity and anti-telomerase properties of yeast Pif1p in vitro.
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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Article: Characterization of the helicase activity and anti-telomerase properties of yeast Pif1p in vitro.
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ABSTRACT: Helicases are ubiquitous enzymes found in all organisms that are necessary for all (or virtually all) aspects of nucleic acid metabolism. The Pif1 helicase family is a group of 5'-->3' directed, ATP-dependent, super family IB helicases found in nearly all eukaryotes. Here, we review the discovery, evolution, and what is currently known about these enzymes in Saccharomyces cerevisiae (ScPif1 and ScRrm3), Schizosaccharomyces pombe (SpPfh1), Trypanosoma brucei (TbPIF1, 2, 5, and 8), mice (mPif1), and humans (hPif1). Pif1 helicases variously affect telomeric, ribosomal, and mitochondrial DNA replication, as well as Okazaki fragment maturation, and in at least some cases affect these processes by using their helicase activity to disrupt stable nucleoprotein complexes. While the functions of these enzymes vary within and between organisms, it is evident that Pif1 family helicases are crucial for both nuclear and mitochondrial genome maintenance.DNA repair 03/2010; 9(3):237-49. DOI:10.1016/j.dnarep.2010.01.008 · 3.36 Impact Factor
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ABSTRACT: Telomeres protect the ends of eukaryotic chromosomes from being recognized and processed as double strand breaks. In most organisms, telomeric DNA is highly repetitive with a high GC-content. Moreover, the G residues are concentrated in the strand running 3'-5' from the end of the chromosome towards its center. This G-rich strand is extended to form a 3' single-stranded tail that can form unusual secondary structures such as T-loops and G-quadruplex DNA. Both the duplex repeats and the single-stranded G-tail are assembled into stable protein-DNA complexes. The unique architecture, high GC content, and multi-protein association create particularly stable protein-DNA complexes that are a challenge for replication, recombination, and transcription. Helicases utilize the energy of nucleotide hydrolysis to unwind base paired nucleic acids and, in some cases, to displace proteins from them. The telomeric functions of helicases from the RecQ, Pifl, FANCJ, and DNA2 families are reviewed in this article. We summarize data showing that perturbation of their telomere activities can lead to telomere dysfunction and genome instability and in some cases human disease.FEBS letters 09/2010; 584(17):3760-72. DOI:10.1016/j.febslet.2010.07.007 · 3.34 Impact Factor
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ABSTRACT: Helicases move on DNA via an ATP binding and hydrolysis mechanism coordinated by well-characterized helicase motifs. However, the translocation along single-stranded DNA (ssDNA) and the strand separation of double-stranded (dsDNA) may be loosely or tightly coupled. Dda is a phage T4 SF1B helicase with sequence homology to the Pif1 family of helicases that tightly couples translocation to strand separation. The crystal structure of the Dda-ssDNA binary complex reveals a domain referred to as the "pin" that was previously thought to remain static during strand separation. The pin contains a conserved phenylalanine that mediates a transient base-stacking interaction that is absolutely required for separation of dsDNA. The pin is secured at its tip by protein-protein interactions through an extended SH3 domain thereby creating a rigid strut. The conserved interface between the pin and the SH3 domain provides the mechanism for tight coupling of translocation to strand separation.Structure 05/2012; 20(7):1189-200. DOI:10.1016/j.str.2012.04.013 · 6.79 Impact Factor