Visualizing helicases unwinding DNA at the single molecule level.

Fili, Natalia, Mashanov, Gregory I., Toseland, Christopher P. , Batters, Christopher, Wallace, Mark I., Yeeles, Joseph T. P., Dillingham, Mark S., Webb, Martin R. and Molloy, Justin E. (2010) Visualizing helicases unwinding DNA at the single molecule level. Nucleic Acids Research, 38 (13). pp. 48-57. ISSN 0305-1048

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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.

Keywords:helicase, DNA unwinding, Single molecule, TIRF
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:46363
Deposited On:10 Jan 2023 13:36

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