Book of Abstracts: Albany 2005
Monitoring Thymine Dimer Repair Electrically
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
Maria C. DeRosa1,*
1California Institute of Technology