Abstract
We describe a novel fluorescence-based assay for detecting DNA conformational alterations within enzyme-DNA complexes. The target adenine for EcoRI DNA methyltransferase (GAATTC) was replaced with 2-aminopurine, which fluoresces upon excitation at 310 nm. Addition of the methyltransferase to the duplex binding site results in a 14-fold increase in fluorescence intensity with a 10 nm blue shift. The fluorescence is approximately 50% of that observed with equimolar free nucleoside, consistent with extrahelical stabilization of the target base in the enzyme-DNA complex. The shift in lambda max further implies the base is placed into a low dielectric environment. For adenine-specific DNA methyltransferases, a hydrophobic pocket composed of highly conserved amino acids lies proximal to the cofactor binding site. Substitution of 2-aminopurine adjacent to the target base also results in detectable changes in fluorescence emission following complex formation with the methyltransferase. Thus, other classes of enzymes hypothesized to utilize base flipping can be investigated by this method.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.