A light-harvesting catalytic DNA for thymine dimer repair

Since the discovery of ribozymes in the 198O's, there have been many examples of catalytic DNA or RNA that can catalyze a variety of reactions, including phosphodiester bond cleavage, porphyrin metallation and nucleotide synthesis. A novel catalytic DNA (DNAzyme) was generated by in vitro selection that could perform a photochemical reaction, the photoreversal of thymine dimers upon irradiation of light. Photolyase enzymes found in nature catalyse the photorepair of thymine dimers with the aid of sensitizers, such as FADH and tryptophan. Curiously, one DNAzyme, UVlC, could repair thymine dimers without the aid of photosensitizers. UV 1 C was found to be active when using light that was significantly red-shifted relative to that of normal DNA absorbance. Using spectroscopic techniques, it was determined that the red-shifted action spectrum and rate enhancement profile were caused by the presence of a guanine quadruplex in the catalytic core. Further characterization of UV1 C revealed interesting information on the folding, substrate specificity and enzyme mechanism. Using sequence mutant constructs, it was determined that the 3'-binding arm of the enzyme could be removed with very little effect on repair rates. Cross-linking reactions determined that the thymine dimer was situated in close proximity, or stacked with the putative guanine quartet. Interestingly, substrates containing uracil dimers with deoxyribose sugar rings were efficiently repaired by UVl C, whereas thymine dimer substrates containing ribosyl sugar rings were not. Oxidative damage to guanines was not detected, which implied that a fast rate of back electron transfer may have occured after the thymine dimer photosplitting process. DNA "aptamers" (binders) were generated by in vitro selection that could simultaneously bind to the electron transfer protein cytochrome c, and to the small metalloporphyrin, hemin. Aptarners selected contained guanine-rich sequences located centrally with respect to the overall sequence. Chemical probing analysis determined that a central guanine-quadruplex formed that was responsible for binding to hemin. Through footprinting analysis, the binding site for cytochrome c on the DNA was determined. The ternary complex represents a potential model for the study of electron transfer between artificial and naturally-occurring heme electron transfer systems.