A fluorescence-based assay for monitoring helicase activity.
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.
Raney
K D
KD
Department of Chemistry, Pennsylvania State University, University Park 16802.
Sowers
L C
LC
Millar
D P
DP
Benkovic
S J
SJ
D015075:2-Aminopurine / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology; D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004265:DNA Helicases / Q000032:analysis / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006860:Hydrogen Bonding; D007700:Kinetics; D008433:Mathematics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D015075:2-Aminopurine; D004247:DNA; D003850:Deoxyribonuclease I; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4