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 suggested 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 photolyase which is >50% identical to E. coli photolyase over the region comprising 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- as well as the band of positive electrostatic potential which defines the DNA binding surface. We found that alanine substitution mutations at sites within the cavity reduced both substrate binding and discrimination, providing direct support for the dinucleotide flip model. The roles of three residues predicted to interact with DNA flanking the dimer were also tested. Arg452 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 structural model for photolyase-dimer interaction is presented
