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

Fili, Natalia and Toseland, Christopher P and Dillingham, Mark S and Webb, Martin R and Molloy, Justin E (2011) A single-molecule approach to visualize the unwinding activity of DNA helicases. Methods in molecular biology (Clifton, N.J.), 778 . pp. 193-214. ISSN 1940-6029. (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

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Abstract

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.

Item Type: Article
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Chris Toseland
Date Deposited: 07 Apr 2015 11:54 UTC
Last Modified: 07 Apr 2015 11:54 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/47853 (The current URI for this page, for reference purposes)
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