Role of Substrate Binding Interactions on DNA Repair by Photolyase

The repair of the cyclobutane pyrimidine dimer (CPD) lesion in DNA by photolyase is determined by its initial recognition, and the catalytic efficiency depends on a series of intermolecular electron-transfer (ET) processes. Here, we investigated the repair of a CPD structural isomer, replacing the deoxyribose with a pyranose sugar on the 5' site, and found a loss in binding efficiency and repair quantum yield. Using femtosecond spectroscopy, we characterized all elementary repair steps and observed a systemic slowdown of the four intermolecular ET reactions and the second bond splitting. Our observations and molecular dynamics simulations suggest that the sugar replacement disrupts the lesion binding configuration, weakening the electronic coupling between the cofactor and lesion and altering the stability of lesion intermediates. These findings highlight how the CPD photolyases have utilized the structural features of the CPD lesion and optimized its interactions with the cofactor and key active-site residues to maximize repair yields.