A fluorescence-based assay for monitoring helicase activity.

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 flourescence-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.

authors

Raney KD,Sowers LC,Millar DP,Benkovic SJ

doi

10.1073/pnas.91.14.6644

subject

Has Abstract

pub_date

1994-07-05 00:00:00

pages

6644-8

issue

14

eissn

0027-8424

issn

1091-6490

journal_volume

91

pub_type

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