Characterization of the cis-syn Thymine Dimer Lesion in Duplex DNA with Nuclear Magnetic Resonance
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Ultraviolet irradiation from sun exposure can lead to permanent, covalent damage in DNA. The damaged sites can block the progression of polymerases, thereby preventing replication or transcription. The frequent occurrence of lesions in the genome necessitates rapid and effective internal repair mechanisms, as well as a highly sensitive detection mechanism to initiate repair. Perturbations in the three-dimensional solution-state structure of the DNA duplex may alert repair proteins to a damage site. Our experiments model the occurrence of a cis-syn thymine cyclobutane dimer lesion, hereafter called the thymine dimer, which results as the major photoproduct of UV irradiation of two adjacent thymines. The two bases form a cyclobutane ring via a [2+2] cycloaddition. The thymine dimer has been considered a bulky and destabilizing lesion; published structures of the thymine dimer lesion reveal that the backbone around the lesion is pinched, the DNA is kinked, and the canonical Watson Crick base pairing with the opposite adenines is disrupted through the loss of one hydrogen bond, weakening the interactions with the complementary strand. We hypothesize that the hydrogen bond disruption from the formation of the lesion results in an increased rate of imino proton exchange from the affected thymine bases. We further predict that an increased rate of proton exchange would correlate to an increased rate at which the bases spontaneously flip out of the double helix conformation, alerting repair enzymes to a damage site. Two-dimensional NMR was used to characterize the imino region of a 10-mer and 12-mer undamaged parent complexes and dimer-containing lesion complexes. Assignment of resonances to specific exchangeable base protons was accomplished through sequential connection of Nuclear Overhauser Effect (NOE) cross-peaks, which result from through-space (rather than through-bond) interactions between protons that are less than ~ 6Å apart. In comparing the imino region of the parent and dimer 12-mer duplex spectra, we observed that the thymines associated with the dimer (T6 and T7) exhibit noticeable upfield shifts in resonance frequency. The thymine at the 5’ end of the dimer (T6) is isolated and does not overlap with neighboring peaks. However, in both the parent and dimer sequences, the thymine residue on the 3’ side of the lesion, T7, is overlaid with a second peak. In the parent, thymines 7 and 9 overlap, and in the dimer, thymine 7 and guanine 14 are overlaid. The position of T7 is not ideal, especially compared with the clarity of T6. Since T7 overlaps with other peaks, we cannot accurately observe changes in behavior once the dimer is introduced. In the 10-mer duplex, a similar upfield shifting effect was observed, but every residue (including dimer residues T5 and T6) is well resolved in both parent and dimer structures. Thus, the 10-mer sequence was used to measure quantitatively the kinetic destabilization using imino exchange experiments. From the exchange experiments, the 5’ thymine dimer residue T5 had an equilibrium constant of opening of 0.11 ×10-6 M-1s-1 in the parent complex, and increased more than tenfold to 1.4 ×10-6 M-1s-1 in the dimer form. Significant increases in equilibrium constants of opening were also observed for the flanking thymine residues T14 and T17, with a modest increase in T6. The increase in equilibrium opening constant for T5 indicates that there is a kinetic effect of the dimer that allows rapid, frequent exchange of the imino proton. Our data support the suggestion that the cis-syn cyclobutane thymine dimer destabilizes the DNA duplex enough to augment base opening to the solvent significantly.