6MAP,a Fluorescent Adenine Analogue,Is a Probe of Base Flipping by DNA Photolyase

摘要

Cyclobutylpyrimidine dimers(CPDs)are formed between adjacent pyrimidines in DNA when it absorbs ultraviolet light.CPDs can be directly repaired by DNA photolyase(PL)in the presence of visible light.How PL recognizes and binds its substrate is still not well understood.Fluorescent nucleic acid base analogues are powerful probes of DNA structure.We have used the fluorescent adenine analogue 6MAP,a pteridone,to probe the local double helical structure of the CPD substrate when bound by photolyase.Duplex melting temperatures were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and the CPD on DNA stability.Steady-state fluorescence measurements of 6MAP-containing single-stranded and doubled-stranded oligos with and without protein show that the local region around the CPD is significantly disrupted.6MAP shows a different quenching pattern compared to 2-aminopurine,another important adenine analogue,although both probes show mat the structure of the complementary strand opposing the 5'-side of the CPD lesion is more destacked than that opposing the 3'-side in substrate/protein complexes.We also show that 6MAP/CPD duplexes are substrates for PL.Vertical excitation energies and transition dipole moment directions for 6MAP were calculated using time-dependent density functional theory.Using these results,the Forster resonance energy transfer efficiency between the individual adenine analogues and the oxidized flavin cofactor was calculated to account for the observed intensity pattern.These calculations suggest that energy transfer is highly efficient for the 6MAP probe and less so for the 2Ap probe.However,no experimental evidence for this process was observed in the steady-state emission spectra.