Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: A study using fluorescent oligonucleotides and fluorescencepolarization

Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K-D values in th e low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K-D determination. Displacement titration is the method of choice; art ifacts due to any direct interaction of the enzyme with the dye are elimina ted, and higher fluorescent-labeled oligonucleotide concentrations may be u sed, improving signal-to-noise ratio. Using this approach (with three diffe rent oligonucleotides) we found that the EcoRV restriction endonuclease sho wed a preference of between 1.5 and 6.5 for its GATATC target sequence at p H 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected , both the presence of Ca2+ and a decrease in pH value stimulated the bindi ng of specific sequences but had much less effect on nonspecific ones.