PMID: 8614617Apr 1, 1996Paper

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands

Nucleic Acids Research
A K EgglestonS C Kowalczykowski

Abstract

We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.

References

Jun 1, 1976·European Journal of Biochemistry·M Abdel-MonemH Hoffmann-Berling
Jan 1, 1979·Cold Spring Harbor Symposia on Quantitative Biology·M DuguetM Gefter
Jan 1, 1979·Cold Spring Harbor Symposia on Quantitative Biology·B KuhnH Hoffmann-Berling
Aug 10, 1979·Nucleic Acids Research·J Kapuściński, W Szer
Jan 1, 1976·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·S A Latt, G Stetten
Aug 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·P P StepienR A Butow
Aug 1, 1992·Molecular Microbiology·J J Lin, A Sancar
Dec 11, 1992·Proceedings of the National Academy of Sciences of the United States of America·T L RipmasterJ L Woolford
Feb 1, 1992·Molecular Microbiology·S R Schmid, P Linder
Jan 1, 1991·Progress in Nucleic Acid Research and Molecular Biology·S W Matson
Jul 26, 1991·Cell·D A Dixon, S C Kowalczykowski
Apr 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·L J RomanS C Kowalczykowski
Sep 1, 1990·Mutation Research·L Grossman, A T Yeung
Jan 1, 1990·Annual Review of Biochemistry·S W Matson, K A Kaiser-Rogers
Aug 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·G T RunyonT M Lohman
Mar 1, 1990·Molecular and Cellular Biology·F RozenN Sonenberg
Jan 16, 1984·European Journal of Biochemistry·I BäumelK Geider
Nov 1, 1980·Cell·A Taylor, G R Smith
Jan 1, 1983·Methods in Enzymology·J Messing
Dec 20, 1983·Nucleic Acids Research·G ManziniF Quadrifoglio
Jul 5, 1994·Proceedings of the National Academy of Sciences of the United States of America·K D RaneyS J Benkovic
Jun 7, 1994·Proceedings of the National Academy of Sciences of the United States of America·P Houston, T Kodadek

❮ Previous
Next ❯

Citations

Aug 4, 2007·Journal of Fluorescence·Cuiling XuPiero R Bianco
Apr 17, 2008·Nature Reviews. Molecular Cell Biology·Timothy M LohmanColin G Wu
Jan 9, 2009·The Journal of Biological Chemistry·Joseph T P YeelesMark S Dillingham
Sep 5, 2003·Nucleic Acids Research·Gökhan Tolun, Richard S Myers
May 30, 1998·Genes & Development·F G Harmon, S C Kowalczykowski
Aug 3, 2010·Journal of Bacteriology·Stephan KolkenbrockSusanne Fetzner
Dec 5, 2008·Microbiology and Molecular Biology Reviews : MMBR·Mark S Dillingham, Stephen C Kowalczykowski
Oct 16, 2008·BioTechniques·Craig A Belon, David N Frick
May 25, 2001·Analytical Biochemistry·L ZhangR K Harrison
May 30, 1998·Proceedings of the National Academy of Sciences of the United States of America·T SugiyamaS C Kowalczykowski
Mar 19, 2014·Extremophiles : Life Under Extreme Conditions·Bang Phuong PhamGang-Won Cheong
Dec 10, 1996·Proceedings of the National Academy of Sciences of the United States of America·F DongP H von Hippel
Mar 31, 1998·Journal of Pharmaceutical Sciences·C R MiddaughD R Casimiro
Nov 6, 2015·Proceedings of the National Academy of Sciences of the United States of America·Behzad RadStephen C Kowalczykowski
May 11, 2010·Methods : a Companion to Methods in Enzymology·Shuo-Xing Dou, Xu Guang Xi
Feb 20, 2010·Methods : a Companion to Methods in Enzymology·Christopher P Toseland, Martin R Webb
Mar 2, 2010·Methods : a Companion to Methods in Enzymology·Yuliang WuRobert M Brosh
Nov 25, 2003·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Peter H von Hippel, Emmanuelle Delagoutte
Nov 18, 2014·Small·Benjamin GollnickFernando Moreno-Herrero
May 29, 2004·Journal of Molecular Biology·Aaron L LuciusTimothy M Lohman
Aug 5, 2006·Journal of Molecular Biology·Barbara DziegielewskaPiero R Bianco
Aug 5, 2006·Bioorganic Chemistry·M Thompson
Jun 11, 2015·The Journal of Physical Chemistry. B·Ashok Pabbathi, Anunay Samanta
Mar 25, 2011·Biochemical Society Transactions·Mark S Dillingham
Jan 29, 2013·RNA Biology·Sebastian L B KönigDavid Rueda
Feb 16, 2013·Enzyme and Microbial Technology·Sammer SiddiquiSyed Ali
May 3, 2016·Methods : a Companion to Methods in Enzymology·Máté GyimesiMihály Kovács
Jul 17, 2004·Organic Letters·Binh T NguyenEric V Anslyn
Mar 13, 1999·Journal of Molecular Biology·E N Zaitsev, S C Kowalczykowski
Sep 15, 1998·Journal of Molecular Biology·D G Anderson, S C Kowalczykowski
Jan 23, 1999·The Journal of Biological Chemistry·E M ZaitsevaS C Kowalczykowski
Oct 12, 2000·The Journal of Biological Chemistry·F G Harmon, S C Kowalczykowski
Jul 26, 2005·The Journal of Biological Chemistry·Mark S DillinghamStephen C Kowalczykowski
Jul 26, 2005·The Journal of Biological Chemistry·Maria SpiesStephen C Kowalczykowski
May 4, 2006·The Journal of Biological Chemistry·Smita S Patel, Ilker Donmez

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Proceedings of the National Academy of Sciences of the United States of America
P Houston, T Kodadek
Proceedings of the National Academy of Sciences of the United States of America
K D RaneyS J Benkovic
© 2021 Meta ULC. All rights reserved