Work

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

Year: 1994

Type: article

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.

Source: Proceedings of the National Academy of Sciences

DOI: 10.1073/pnas.91.12.5471

Authors: P Houston, T Kodadek

Institution: The University of Texas at Austin

Language: English

Topic: DNA and Nucleic Acid Chemistry

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: National Institutes of Health, National Institute of General Medical Sciences

Awards: G145969878, BIOCHEMISTRY OF T4 HOMOLOGOUS RECOMBINATION