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. 1994 Jun 7;91(12):5471–5474. doi: 10.1073/pnas.91.12.5471

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

P Houston 1, T Kodadek 1
PMCID: PMC44017  PMID: 8202510

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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