A fluorescence-based assay for monitoring helicase activity.
- 5 July 1994
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 91 (14) , 6644-6648
- https://doi.org/10.1073/pnas.91.14.6644
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.Keywords
This publication has 16 references indexed in Scilit:
- Influence of 5'-nearest neighbors on the insertion kinetics of the fluorescent nucleotide analog 2-aminopurine by Klenow fragmentBiochemistry, 1993
- Overexpression, purification, sequence analysis, and characterization of the T4 bacteriophage dda DNA helicase.Journal of Biological Chemistry, 1992
- Strand-Specific Recognition of a Synthetic DNA Replication Fork by the SV40 Large Tumor AntigenScience, 1992
- The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNABiochemistry, 1992
- Allosteric Effects of Nucleotide Cofactors on Escherichia coli Rep Helicase&DNA BindingScience, 1992
- Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamerBiochemistry, 1992
- Dynamics of mismatched base pairs in DNABiochemistry, 1991
- DNA HELICASESAnnual Review of Biochemistry, 1990
- Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studiesBiochemistry, 1989
- Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR.Proceedings of the National Academy of Sciences, 1986