Single-Molecule Fluorescence Reveals the Unwinding Stepping Mechanism of Replicative Helicase

Salman Syed, Manjula Pandey, Smita S. Patel, Taekjip Ha

Research output: Contribution to journalArticle

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 2-3bp 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.

Original languageEnglish (US)
Pages (from-to)1037-1045
Number of pages9
JournalCell Reports
Volume6
Issue number6
DOIs
StatePublished - Mar 27 2014
Externally publishedYes

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Fluorescence
Base Pairing
Hydrolysis
Molecules
Cytosine
Guanine
DNA
Bacteriophage T7
Fluorescence Resonance Energy Transfer
Bacteriophages
Nucleic Acids
thymidine 5'-triphosphate
Enzymes
Proteins

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Single-Molecule Fluorescence Reveals the Unwinding Stepping Mechanism of Replicative Helicase. / Syed, Salman; Pandey, Manjula; Patel, Smita S.; Ha, Taekjip.

In: Cell Reports, Vol. 6, No. 6, 27.03.2014, p. 1037-1045.

Research output: Contribution to journalArticle

Syed, Salman ; Pandey, Manjula ; Patel, Smita S. ; Ha, Taekjip. / Single-Molecule Fluorescence Reveals the Unwinding Stepping Mechanism of Replicative Helicase. In: Cell Reports. 2014 ; Vol. 6, No. 6. pp. 1037-1045.
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