title: Yeast Pif1 helicase exhibits a one-base-pair stepping mechanism for unwinding duplex DNA.
abstract: Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
authors.forename: Ramanagouda
authors.initials: R
authors.lastname: Ramanagoudr-Bhojappa
authors.forename: Shubeena
authors.initials: S
authors.lastname: Chib
authors.forename: Alicia K
authors.initials: AK
authors.lastname: Byrd
authors.forename: Suja
authors.initials: S
authors.lastname: Aarattuthodiyil
authors.forename: Manjula
authors.initials: M
authors.lastname: Pandey
authors.forename: Smita S
authors.initials: SS
authors.lastname: Patel
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Raney
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C085463:PIF1 protein, S cerevisiae; D004265:DNA Helicases
keywords: DNA Helicase; DNA Unwinding; DNA-binding Protein; Kinetics; Protein Displacement; Protein Translocation; Step Size; Yeast