Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer.
The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation. The steady state kinetic parameters for these substrates were in agreement with the rate constants obtained from a gel electrophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sensitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.
Ghosh
S S
SS
Life Sciences Research Laboratory, Baxter Diagnostics, Inc., San Diego, CA 92121.
Eis
P S
PS
Blumeyer
K
K
Fearon
K
K
Millar
D P
DP
D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D019793:Fluorescein; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D012235:Rhodamines; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D019793:Fluorescein