A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands

Angela K. Eggleston, Nazir A. Rahim, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1179-1186
Number of pages8
JournalNucleic Acids Research
Volume24
Issue number7
DOIs
StatePublished - 1996

Fingerprint

Nucleic Acids
Exodeoxyribonuclease V
Coloring Agents
Ligands
DNA
RNA Helicases
DNA Helicases
Bisbenzimidazole
Acridine Orange
Ethidium
Fluorescent Dyes
Salts
Fluorescence
Escherichia coli
Temperature
Enzymes

ASJC Scopus subject areas

  • Genetics

Cite this

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands. / Eggleston, Angela K.; Rahim, Nazir A.; Kowalczykowski, Stephen C.

In: Nucleic Acids Research, Vol. 24, No. 7, 1996, p. 1179-1186.

Research output: Contribution to journalArticle

Eggleston, Angela K. ; Rahim, Nazir A. ; Kowalczykowski, Stephen C. / A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands. In: Nucleic Acids Research. 1996 ; Vol. 24, No. 7. pp. 1179-1186.
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