A fluorescence-based assay for monitoring helicase activity

Kevin D. Raney, Lawrence C. Sowers, David P. Millar, Stephen J. Benkovic

Research output: Contribution to journalArticlepeer-review

120 Scopus citations

Abstract

A continuous fluorescence-based assay is described for measuring helicase- mediated unwinding of duplex DNA. The assay utilizes an oligonucleotide substrate containing the fluorescent adenine analog, 2-aminopurine, at regular intervals. 2-Aminopurine forms a Watson-Crick-type base pair with thymine and does not distort normal B-form DNA. Fluorescence of the 2- aminopurines within this oligonucleotide is quenched 2-fold upon its hybridization to a complementary strand. Unwinding of this substrate by the T4 dda helicase restores the fluorescence of the 2-aminopurines and is easily followed using stopped-flow or steady-state fluorescence spectroscopy. The fluorescence-based assay provides rate data comparable to that obtained from conventional discontinuous assays using labeled substrates and additionally furnishes a means for following a single turnover. This assay should prove useful for defining the mechanism by which helicases unwind duplex DNA.

Original languageEnglish (US)
Pages (from-to)6644-6648
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number14
DOIs
StatePublished - Jul 5 1994

All Science Journal Classification (ASJC) codes

  • General

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