DNA-modified gold electrodes exploit both the ability of DNA to act as an efficient conduit for charge transport (CT) as well as its exquisite sensitivity to any perturbations in the π-stacking of the DNA base pairs. These modified surfaces serve as versatile platforms for electrochemical studies of DNA-protein interactions such as restriction and base-flipping (1). Photolyase is a light-activated flavo-enzyme that repairs cyclobutane pyrimidine dimer (CPD) lesions in DNA in a reaction triggered by electron transfer from a photoexcited flavin cofactor to the CPD (2). Biochemical and structural data suggest that the CPD is flipped out of the DNA helix into a pocket containing the catalytic flavin cofactor (2, 3). Using gold electrodes modified with DNA duplexes containing a thymine dimer (T<>T), here we probe the electrochemistry of the flavin in E. coli photolyase during the DNA repair process.

Cyclic and square wave voltammetry of photolyase deposited on gold electrodes show a redox signal at -160 mV vs. Ag/AgCl, consistent with electron transfer to and from the flavin cofactor in the DNA-bound protein. This signal is not observed when photolyase is incubated on surfaces modified with undamaged DNA or simply mercaptohexanol. Furthermore, the signal is drastically attentuated on surfaces where the π-stacking of the DNA bases is perturbed by the presence of an abasic site below the thymine dimer, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically; irradiation of photolyase on T<>T damaged DNA films leads to changes in the peak current which track with changes in the structure of the DNA duplex as the T<>T lesion is repaired. The flipped-out T<>T lesion attenuates CT from the electrode to the flavin cofactor, leading initially to a weak signal. Upon repair, however, signal intensity increases as the restored DNA π-stack allows for efficient DNA-mediated communication between the gold surface and the flavin.


References and Footnotes

1. Boon, E. M., Salas, J. E., and Barton, J. K. Nature Biotech. 20, 282-286 (2002).
2. Sancar, A. Chem. Rev. 103, 2203-2237 (2003).
3. Mees, A., Klar, T., Gnau, P., Hennecke, U., Eker, A. P. M., Carell, T., and Essen, L. O. Science 306, 17889-1793 (2004).