NMR and thermodynamic studies on photoproduct-containing DNA dodecamers

Cis-syn cyclobutane photodimers, (6-4) pyrimidine-pyrimidone photoproducts, and their Dewar valence isomers are the major photoproducts of DNA and have different enzymatic repair and mutagenic properties. To begin to understand the underlying physical basis for these properties, NMR and melting temperature studies were carried out on a series of photoproduct-containing DNA duplexes. In one study, the structure of the dodecamer duplex d(GCACGAAT[cis-syn]TAAG) .d(CTTAATTCGTGC) corresponding to the binding site of the cis-syn cyclobutane dimer-specific T4 endonuclease V repair enzyme on a site-specific dimer-containing 49-mer was investigated by NMR. Proton and 31P NMR resonances of the cis-syn thymine dimer-containing dodecamer duplex and the undamaged parental duplex were assigned. The unusually shifted T8-NH3 imino, A16-H2, T8-Me proton resonances and the T9pA10 31P NMR resonance of the dimer-containing duplex could be explained on the basis of three dimensional models that were derived from NOE restrained molecular dynamics and simulated annealing calculations. The structure revealed the presence of a BII-type phosphodiester linkage flanking the 3' -site of the dimer, which could explain how it is recognized by the T4 endonuclease. In a second study, the thermal stability and base pairing properties of the cis-syn, (6-4), and Dewar photoproducts of the TT site in d(GAGTATTATGAG) were also investigated. The (6-4) and Dewar photoproducts were found to destabilize the duplex form by about 6 kcal/mole of free energy at 37°C relative to the parent duplex, whereas the cis-syn dimer was only found to destabilize the duplex form by 1.5 kcal/mol. Duplexes with G opposite the 3' -T of the (6-4) and Dewar products were more stable than those with A by about 0.4 kcal/mol, whereas the cis-syn dimer preferred A over G by 0.7 kcal/mol. Proton NMR studies suggest that wobble base pairing takes place between the 3'-T of the cis-syn dimer and an opposed G, whereas there is no evidence of significant H-bonding between these two bases in the (6-4) product. The thermodynamic and H-bonding data for the (6-4) product are consistent with a four nucleotide interior loop structure which may facilitate flipping of the photoproduct in and out of the helix and explain how it is recognized by the (6-4) photolyase repair enzyme. In general, the relative rates of excision repair of the photoproducts were found to parallel the thermodynamic data best, with the exception of a cis-syn thymine dimer with a G opposite the 3'-T.