A FLUORESCENCE-BASED ASSAY FOR MONITORING HELICASE ACTIVITY

A continuous fluorescence-based assay is described for measuring helic ase-mediated unwinding of duplex DNA. The assay utilizes an oligonucle otide substrate containing the fluorescent adenine analog, 2-aminopuri ne, at regular intervals. 2-Aminopurine forms a Watson-Crick type base pair with thymine and does not distort normal B-form DNA. Fluorescenc e of the 2-aminopurines within this oligonucleotide is quenched 2-fold upon its hybridization to a complementary strand. Unwinding of this s ubstrate by the T4 dda helicase restores the fluorescence of the 2-ami nopurines and is easily followed using stopped-now or steady-state flu orescence spectroscopy. The fluorescence-based assay provides rate dat a comparable to that obtained from conventional discontinuous assays u sing labeled substrates and additionally furnishes a means for followi ng a single turnover. This assay should prove useful for defining the mechanism by which helicases unwind duplex DNA.