Spectrophotometric assay for enzyme-mediated unwinding of double-stranded DNA.

Abstract:

:A method is described for monitoring the enzyme-mediated unwinding of duplex DNA spectrophotometrically. The assay employs a partially duplex oligonucleotide substrate modified at the complementary end with coumarin and fluorescein moieties. When in close proximity the fluorescein quenches the fluorescence of coumarin. However, when the strands are separated by the action of a DNA helicase, the coumarin fluorescence increases greatly. Therefore, the progress of enzyme-mediated DNA unwinding can be measured in real time by fluorescence spectroscopy. This assay provides a simple method to screen for helicase inhibitors, which are of growing interest as potential anticancer agents. The application of this technique to kinetic analyses of the mechanism of action of DNA helicases is also discussed.

authors

Houston P,Kodadek T

doi

10.1073/pnas.91.12.5471

subject

Has Abstract

pub_date

1994-06-07 00:00:00

pages

5471-4

issue

12

eissn

0027-8424

issn

1091-6490

journal_volume

91

pub_type

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