Abstract
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.
Highlights
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
Where the underlined A indicates the position of the 2-aminopurine base in the 2-AP containing ss-DNA, T-T stands for the complementary strand, and TϽϾT indicates the cyclobutylthymidine dimer-containing oligonucleotide
UV-A lamps were used to generate the Cyclobutylpyrimidine dimers (CPDs) lesion in the TT-containing 11-mer oligonucleotide, and HPLC purification of the photoproduct was performed as described by Banerjee et al [26]
Summary
DNA methyltransferase is thought to base flip an adenine into the enzyme for methylation [15, 16] Another repair protein, T4 endonuclease V, has been shown to flip an adenine opposing the CPD lesion out of the helix [17]. T4 endonuclease V, has been shown to flip an adenine opposing the CPD lesion out of the helix [17] Several of these studies have used a fluorescent adenine analog, 2-aminopurine (2-Ap), as a reporter of helical structure. The quantum yield and emission maximum of 2-Ap are effective reporters of base stacking and solvent exposure (polarity), respectively We have utilized these properties to probe base flipping in photolyase by incorporated 2-Ap opposite a thymidine dimer.
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