TARGETED BASE STACKING DISRUPTION BY THE ECORI DNA METHYLTRANSFERASE

We describe a novel fluorescence-based assay for detecting DNA conform ational alterations within enzyme-DNA complexes. The target adenine fo r EcoRI DNA methyltransferase (GA(A)TTC) was replaced with 2-aminopuri ne, which fluoresces upon excitation at 310 nm. Addition of the methyl transferase to the duplex binding site results in a 14-fold increase i n fluorescence intensity with a 10 nm blue shift. The fluorescence is similar to 50% of that observed with equimolar free nucleoside, consis tent with extrahelical stabilization of the target base in the enzyme- DNA complex. The shift in lambda(max) further implies the base is plac ed into a low dielectric environment. For adenine-specific DNA methylt ransferases, a hydrophobic pocket composed of highly conserved amino a cids lies proximal to the cofactor binding site. Substitution of 2-ami nopurine adjacent to the target base also results in detectable change s in fluorescence emission following complex formation with the methyl transferase. Thus, other classes of enzymes hypothesized to utilize ba se flipping can be investigated by this method.