REAL-TIME KINETICS OF RESTRICTION-ENDONUCLEASE CLEAVAGE MONITORED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER

The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of flu orophor-labeled oligonucleotide substrates have been examined using fl uorescence resonance energy transfer (FRET). A series of duplex substr ates were synthesized with an internal CTCGAG PaeR7 recognition site a nd donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescenc e resulting from restriction cleavage of these substrates was continuo usly monitored and the initial rate data was fitted to the Michaelis-M enten equation. The steady state kinetic parameters for these substrat es were in agreement with the rate constants obtained from a gel elect rophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sen sitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.