A Model for the Enzyme−Substrate Complex of DNA Photolyase and Photodamaged DNA

The three-dimensional structure of Escherichia coli DNA photolyase and molecular dynamics simulations using the AMBER force field were used to construct a model of the enzyme−substrate complex. Three different dinucleotides with cyclobutane pyrimidine dimers ( T<>T, T<>U, and U<>T ), two conformations of a single-stranded DNA nonamer, and a duplex DNA dodecamer containing the T<>T lesion were studied. The results are in good agreement with available experimental data and provide a structural rationalization for the results of ethylation studies, the measurement of the relative rates of electron transfer for different dinucleotides complexed to the enzyme, and the similar binding constants for T<>T containing single stranded and duplex DNA. The results support the base-flipping mechanism suggested earlier. The proposed active-site model reveals three types of interactions: (i) ion-pair interactions at the rim of the active site between the positively charged residues on the enzyme surface (Arg 226, Arg 342, Arg 397, and Lys 154 ) and the deoxyribophosphate immediately 5‘ to the dimer as well as the three deoxyribophosphates on the 3‘ side, (ii) polar interactions between Glu 274 and the NH function of the 3‘ base of the dimer as well as a hydrogen bond between the C-4 carbonyl on the 5‘ base of the dimer with Trp 384, and (iii) hydrophobic interactions between Trp 277 and Trp 384 and the nonpolar cyclobutane moiety of the dimer, thus shielding the radical anion intermediate of the DNA repair from electrophilic attack. In this model, the distance between the redox active FADH cofactor and the dimer is too large to account for the observed rates of electron transfer. Rather, the results suggest an electron transfer mediated by the π-systems of the aromatic residues Trp 277 and Trp 384 .