Differential Distortion of Substrate Occurs When It Binds to DNA Photolyase: A 2-Aminopurine Study

Abstract

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it is exposed to ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) upon absorption of blue-green light. We have used the fluorescent adenine analogue 2-aminopurine (2Ap) to probe the local double-helical structure of the DNA substrate when it binds to the protein. Duplex melting temperatures and van't Hoff enthalpies were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and CPD on DNA stability. Steady-state fluorescence measurements of the single- and double-stranded oligos showed that the local region around the 5'-side of the CPD lesion was more disrupted and destacked than the 3'-side in substrate-protein complexes. These results were compared with those of a protein-substrate crystal structure, demonstrating that the crystal structure and solution-state studies are in agreement with regard to the differential distortions of the target DNA at the active site of the protein.