A single-molecule approach to visualize the unwinding activity of DNA helicases.

Fili, Natalia, Toseland, Christopher P, Dillingham, Mark S , Webb, Martin R and Molloy, Justin E (2011) A single-molecule approach to visualize the unwinding activity of DNA helicases. In: Single Molecule Enzymology Methods and Protocols. Springer, pp. 193-214. ISBN 9781493961900

Full content URL: https://doi.org/10.1007/978-1-61779-261-8_13

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Item Type:Book Section
Item Status:Live Archive


Almost all aspects of DNA metabolism involve separation of double-stranded DNA catalyzed by helicases. Observation and measurement of the dynamics of these events at the single-molecule level provide important mechanistic details of helicase activity and give the opportunity to probe aspects that are not revealed in bulk solution measurements. The assay, presented here, provides information about helicase unwinding rates and processivity. Visualization is achieved by using a fluorescent single-stranded DNA-binding protein (SSB), which allows the time course of individual DNA unwinding events to be observed using total internal reflection fluorescence microscopy. Observation of a prototypical helicase, Bacillus subtilis AddAB, shows that the unwinding process consists of bursts of unwinding activity, interspersed with periods of pausing.

Keywords:DNA unwinding, helices assay, Single molecule, protocol
Subjects:C Biological Sciences > C700 Molecular Biology, Biophysics and Biochemistry
Divisions:College of Science > School of Life and Environmental Sciences > Department of Life Sciences
ID Code:46362
Deposited On:16 Jan 2023 14:22

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