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Cell Rep. 2014 Mar 27;6(6):1037-1045. doi: 10.1016/j.celrep.2014.02.022. Epub 2014 Mar 13.

Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase.

Author information

1
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
2
Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
3
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.

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.

PMID:
24630993
PMCID:
PMC3988844
DOI:
10.1016/j.celrep.2014.02.022
[Indexed for MEDLINE]
Free PMC Article

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