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 language | English (US) |
|---|---|
| Pages (from-to) | 1179-1186 |
| Number of pages | 8 |
| Journal | Nucleic Acids Research |
| Volume | 24 |
| Issue number | 7 |
| DOIs | |
| State | Published - 1996 |
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ASJC Scopus subject areas
- Genetics
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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 journal › Article
}
TY - JOUR
T1 - A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands
AU - Eggleston, Angela K.
AU - Rahim, Nazir A.
AU - Kowalczykowski, Stephen C.
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/24.7.1179
DO - 10.1093/nar/24.7.1179
M3 - Article
C2 - 8614617
AN - SCOPUS:0029879214
VL - 24
SP - 1179
EP - 1186
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 7
ER -