title: Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase.
abstract: Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2-3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Salman
authors.initials: S
authors.lastname: Syed
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
authors.forename: Manjula
authors.initials: M
authors.lastname: Pandey
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
authors.forename: Smita S
authors.initials: SS
authors.lastname: Patel
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Howard Hughes Medical Institute, Urbana, IL 61801, USA. Electronic address: tjha@illinois.edu.
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*
chemical_list: D004247:DNA; D004265:DNA Helicases