Visualizing helicases unwinding DNA at the single molecule level.

Fili, Natali and Mashanov, Gregory I and Toseland, Christopher P and Batters, Christopher and Wallace, Mark I and Yeeles, Joseph T P and Dillingham, Mark S and Webb, Martin R and Molloy, Justin E (2010) Visualizing helicases unwinding DNA at the single molecule level. Nucleic acids research, 38 (13). pp. 4448-57. ISSN 1362-4962. (Full text available)

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Abstract

DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.

Item Type: Article
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Chris Toseland
Date Deposited: 07 Apr 2015 11:52 UTC
Last Modified: 07 Apr 2015 12:09 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/47855 (The current URI for this page, for reference purposes)
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