title: A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End.
abstract: Using a DNA polymerase coupled assay and FRET (Förster resonance energy transfer)-based helicase assays, in this work, we show that a monomer of Saccharomyces cerevisiae Pif1 can unwind dsDNA (double-stranded DNA). The helicase activity of a Pif1 monomer is modulated by the nature of the 3'-ssDNA (single-stranded DNA) tail of the substrate and its effect on a Pif1-dependent re-winding activity that is coupled to the opening of dsDNA. We propose that, in addition to the ssDNA site on the protein that interacts with the translocating strand, Pif1 has a second site that binds the 3'-ssDNA of the substrate. Interaction of DNA with this site modulates the degree to which re-winding counteracts unwinding. Depending on the nature of the 3'-tail and the length of the duplex DNA to be unwound, this activity is sufficiently strong to mask the helicase activity of a monomer. In excess Pif1 over the DNA, the Pif1-dependent re-winding of the opened DNA strongly limits unwinding, independent of the 3'-tail. We propose that, in this case, binding of DNA to the second site is precluded and modulation of the Pif1-dependent re-winding activity is largely lost.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors.forename: Saurabh P
authors.initials: SP
authors.lastname: Singh
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors.forename: Katrina N
authors.initials: KN
authors.lastname: Koc
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Stodola
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA. Electronic address: galletto@biochem.wustl.edu.
authors.forename: Roberto
authors.initials: R
authors.lastname: Galletto
mesh_terms: D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C085463:PIF1 protein, S cerevisiae; D004265:DNA Helicases
keywords: FRET; helicase; stopped-flow; strand displacement DNA synthesis