DNA Photolyase is a flavoprotein that uses light to repair cyclobutylpyrimidine dimers in DNA. From considerations of the crystal structure of the protein, it has been hypothesized that the dimer lesion is flipped out of the DNA double helix into the substrate binding pocket. We have used a fluorescent adenine analog, 2-aminopurine (2-Ap), as a probe of local double helical structure upon binding of the substrate to the protein. Our results show that the local structure around the thymidine lesion changes dramatically upon binding to Photolyase. This is consistent with base flipping of the lesion into the protein binding cavity with concomitant destacking of the opposing complementary 2-Ap nucleotide.Cyclobutylpyrimidine dimers (CPDs) 1 are lesions formed in DNA between adjacent pyrimidines upon absorption of UV light. These lesions cause replicational errors and can lead to cell death or cancer if left unrepaired (1, 2). One repair protein, DNA photolyase, incorporates a non-covalently bound FAD and requires blue light for repair (3). There is ample evidence that repair of the CPD proceeds by electron transfer from a photoexcited fully reduced FADH Ϫ to the CPD, which subsequently monomerizes within 2 ns (3). Studies by Payne et al. (4) have demonstrated that the oxidized enzyme can bind CPD-containing DNA but cannot efficiently repair the CPD lesion. As we show below, this differential behavior is extremely useful in understanding the substrate binding mode of photolyase.The crystal structures of the Escherichia coli and Aspergillus nidulans holoenzymes were solved by Kim et al. (5) and Tamada and co-workers (6), respectively. These crystal structures revealed important structural elements that were both familiar and surprising. It was noted that the cavity has approximately the correct dimensions to enclose the CPD if the thymidines were folded in a coplanar geometry, leading to the prediction that photolyase would bind the CPD by "flipping out" the lesion from the double helical DNA (5). The FADH Ϫ cofactor was found to lie at the bottom of this cavity, consistent with the ability of the cofactor to resist oxidation by molecular O 2 . The surface of the protein above the cavity incorporates a strip of positively charged amino acid residues that are thought to help orient the negatively charged phosphate backbone of the damaged DNA strand.Unfortunately, no crystal structure of the enzyme-substrate complex is available, but a recent crystal structure of Thermus thermophilus complexed with thymine was published by Komori and co-workers (7), which shows the thymine residing in the putative substrate cavity. While thymine is not a substrate, it can be thought of as a partial product of the repair reaction. This is the strongest evidence regarding the substrate binding mode of photolyase for CPDs. However, it does not clarify whether one or both of the thymine bases of the CPD is bound in the cavity.Other experimental evidence for the mode of substrate binding is available. Most notable are the ethylation and ...

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.

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 that 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 Förster 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.

Cyclobutylpyrimidine dimers (CPDs) are the major UV photoproduct formed in DNA containing adjacent pyrimidines. These lesions can be repaired by DNA photolyase, a flavoprotein that utilizes blue light in a direct reversal of the cyclobutane ring. Previous studies have shown that the CPD is base flipped into the protein, with concomitant disruption of the substrate around the CPD. In this study, we use a fluorescent cytidine analog, pyrrolo-dC (PC), to probe how far base flipping propagates along the duplex. From these measurements, the degree of base destacking in the two bases flanking the adenines opposing the CPD appears to be minimal, which was consistent with the protein:substrate crystal structure. Fluorescence-detected melting temperatures for duplexes with and without a CPD were obtained, suggesting that a 5'-pyrimidine-PC-purine-3' motif is more stable than the 5'-purine-PC-pyrimidine-3' motif. This stability trend was reflected in the fluorescence intensities of ss-PC oligos but not for duplexes. The melting point depression due to the PC probe was found to be comparable to other popular fluorescent base analogs.

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