Nucleic Acids Research, Vol 24, Issue 7 1179-1186, Copyright © 1996 by Oxford University Press
AK Eggleston, NA Rahim and SC Kowalczykowski
We have developed a new helicase assay that overcomes many limitations of
other assays used to measure this activity. This continuous, kinetic assay
is based on the displacement of fluorescent dyes from dsDNA upon DNA
unwinding. These ligands exhibit significant fluorescence enhancement when
bound to duplex nucleic acids and serve as the reporter molecules of DNA
unwinding. We evaluated the potential of several dyes [acridine orange,
ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258
and thiazole orange] to function as suitable reporter molecules and
demonstrate that the latter three dyes can be used to monitor the helicase
activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry
of RecBCD enzyme for the ends of duplex DNA and the apparent rate of
unwinding are not significantly perturbed by two of these dyes. The effects
of temperature and salt concentration on the rate of unwinding were also
examined. We propose that this dye displacement assay can be readily
adapted for use with other DNA helicases, with RNA helicases, and with
other enzymes that act on nucleic acids.
ARTICLES
A helicase assay based on the displacement of fluorescent, nucleic acid- binding ligands
Division of Biological Sciences, University of California, Davis 95616- 8665, USA.
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