Hexameric helicase G40P unwinds DNA in single base pair steps
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 ATPgS 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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Due to their large size (~200Gb total), raw video data files are available upon request.
Article and author information
Author details
Funding
National Institutes of Health (GM065367)
- Taekjip Ha
National Science Foundation (PHY-082261)
- Taekjip Ha
Deutsche Forschungsgemeinschaft (SCHL1896/1-1)
- Michael Schlierf
Bundesministerium für Bildung und Forschung (03Z2EN11)
- Michael Schlierf
Howard Hughes Medical Institute
- Taekjip Ha
National Institutes of Health (137405)
- Xiaojiang S Chen
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Schlierf et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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