In the absence of a more sophisticated technique that monitors enzyme-DNA binding, DNA structural changes and base flipping simultaneously, the interpretation of the formation of intermediates and catalysis from an enzyme-DNA interaction remains ambiguous. In our lab, we propose to develop single molecule assays to monitor the base flipping mechanism in DNA-enzyme complexes in real time. Base flipping corresponds to the rotation of a DNA nucleotide out of the double helix, and its accommodation into a protein-binding pocket inside the enzyme. Understanding base flipping's biophysical basis is crucial due to its role in DNA repair. The fluorescence properties of 2-amino-6(2-thienyl)purine (Fluorescent Thymine Analog, FTA) will be used to probe base flipping which may be induced from the interaction of an enzyme with a FTA-substituted DNA. The objective of this project is the ensemble fluorescence characterization of different DNA-FTA-modified constructions that will mimic the different states during the base flipping process.

Photophysical characterization of a fluorescent thymine analog to probe base flipping in DNA

243rd National Meeting (2012)

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