DOI: 10.1073/pnas.91.14.6644. A fluorescence-based assay for monitoring helicase activity.

A fluorescence-based assay for monitoring helicase activity.

10.1073/pnas.91.14.6644

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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.

Bibliography

Raney, K. D., Sowers, L. C., Millar, D. P., & Benkovic, S. J. (1994). A fluorescence-based assay for monitoring helicase activity. Proceedings of the National Academy of Sciences, 91(14), 6644â6648.

Authors 4

K D Raney (first)
L C Sowers (additional)
D P Millar (additional)
S J Benkovic (additional)

References 0 Referenced 101

None

Dates

Type	When
Created	19 years, 2 months ago (May 31, 2006, 8:50 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 1:45 p.m.)
Indexed	1 month ago (July 24, 2025, 6:55 a.m.)
Issued	31 years, 1 month ago (July 5, 1994)
Published	31 years, 1 month ago (July 5, 1994)
Published Online	31 years, 1 month ago (July 5, 1994)
Published Print	31 years, 1 month ago (July 5, 1994)

Funders 0

None

BibTeX

@article{Raney_1994, title={A fluorescence-based assay for monitoring helicase activity.}, volume={91}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.91.14.6644}, DOI={10.1073/pnas.91.14.6644}, number={14}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Raney, K D and Sowers, L C and Millar, D P and Benkovic, S J}, year={1994}, month=jul, pages={6644–6648} }

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