A fluorescence-based assay for monitoring helicase activity

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

Research output: Contribution to journalArticle

118 Citations (Scopus)

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
Externally publishedYes

Fingerprint

2-Aminopurine
Fluorescence
Oligonucleotides
B-Form DNA
Thymine
DNA
Fluorescence Spectrometry
Adenine
Base Pairing

Keywords

  • 2-aminopurine
  • dda helicase
  • stopped-flow spectroscopy

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

A fluorescence-based assay for monitoring helicase activity. / Raney, Kevin D.; Sowers, Lawrence; Millar, David P.; Benkovic, Stephen J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 14, 05.07.1994, p. 6644-6648.

Research output: Contribution to journalArticle

Raney, Kevin D. ; Sowers, Lawrence ; Millar, David P. ; Benkovic, Stephen J. / A fluorescence-based assay for monitoring helicase activity. In: Proceedings of the National Academy of Sciences of the United States of America. 1994 ; Vol. 91, No. 14. pp. 6644-6648.
@article{0d287501f0984a759b1aee607c31aca9,
title = "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 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.",
keywords = "2-aminopurine, dda helicase, stopped-flow spectroscopy",
author = "Raney, {Kevin D.} and Lawrence Sowers and Millar, {David P.} and Benkovic, {Stephen J.}",
year = "1994",
month = "7",
day = "5",
doi = "10.1073/pnas.91.14.6644",
language = "English (US)",
volume = "91",
pages = "6644--6648",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "14",

}

TY - JOUR

T1 - A fluorescence-based assay for monitoring helicase activity

AU - Raney, Kevin D.

AU - Sowers, Lawrence

AU - Millar, David P.

AU - Benkovic, Stephen J.

PY - 1994/7/5

Y1 - 1994/7/5

N2 - 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.

AB - 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.

KW - 2-aminopurine

KW - dda helicase

KW - stopped-flow spectroscopy

UR - http://www.scopus.com/inward/record.url?scp=0028224662&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028224662&partnerID=8YFLogxK

U2 - 10.1073/pnas.91.14.6644

DO - 10.1073/pnas.91.14.6644

M3 - Article

VL - 91

SP - 6644

EP - 6648

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 14

ER -