EVIDENCE FOR DINUCLEOTIDE FLIPPING BY DNA PHOTOLYASE

DNA photolyases repair pyrimidine dimers via a reaction in which light energy drives electron donation from a catalytic chromophore, FADH(-) , to the dimer. The crystal structure of Escherichia coli photolyase s uggested that the pyrimidine dimer is flipped out of the DNA helix and into a cavity that leads from the surface of the enzyme to FADH-. We have tested this model using the Saccharomyces cerevisiae Phr1 photoly ase which is >50% identical to E. coli photolyase over the region comp rising the DNA binding domain. By using the bacterial photolyase as a starting point, we modeled the region encompassing amino acids 383-530 of the yeast enzyme. The model retained the cavity leading to FADH- a s well as the band of positive electrostatic potential which defines t he DNA binding surface. We found that alanine substitution mutations a t sites within the cavity reduced both substrate binding and discrimin ation, providing direct support for the dinucleotide flip model. The r oles of three residues predicted to interact with DNA flanking the dim er were also tested. Arg(452) was found to be particularly critical to substrate binding, discrimination, and photolysis, suggesting a role in establishing or maintaining the dimer in the flipped state. A struc tural model for photolyase-dimer interaction is presented.