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Elife. 2019 Jan 28;8. pii: e42001. doi: 10.7554/eLife.42001.

Hexameric helicase G40P unwinds DNA in single base pair steps.

Schlierf M1,2, Wang G3, Chen XS3, Ha T1,4,5,6,7.

Author information

1
Physics Department and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Illinois, United States.
2
B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany.
3
Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United States.
4
Howard Hughes Medical Institute, Baltimore, United States.
5
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, United States.
6
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, United States.
7
Department of Biophysics, Johns Hopkins University, Baltimore, United States.

Abstract

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

KEYWORDS:

DnaB; DnaG; G40P; molecular biophysics; none; single-molecule FRET; structural biology

PMID:
30688211
DOI:
10.7554/eLife.42001
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