The cis-(5R,6S)-Thymine Glycol Lesion Occupies the Wobble Position When Mismatched with Deoxyguanosine in DNA

Abstract

Oxidative damage to 5-methylcytosine in DNA, followed by deamination, yields thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, mispaired with deoxyguanosine. The structure of the 5R Tg·G mismatch pair has been refined using a combination of simulated annealing and isothermal molecular dynamics calculations restrained by NMR-derived distance restraints and torsion angle restraints in 5′-d(G1T2G3C4G5Tg6G7T8T9T10G11T12)-3′·5′-d(A13C14A15A16A17C18G19C20G21C22A23C24)-3′; Tg = 5R Tg. In this duplex the cis-5R,6S:trans-5R,6R equilibrium favors the cis-5R,6S epimer [Brown, K. L., Adams, T., Jasti, V. P., Basu, A. K., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 11701−11710]. The cis-5R,6S Tg lesion is in the wobble orientation such that Tg6O2 is proximate to G19 N1H and Tg6 N3H is proximate to G19O6. Both Tg6 and the mismatched nucleotide G19 remain stacked in the helix. The Tg6 nucleotide shifts toward the major groove and stacks below the 5′-neighbor base G5, while its complement G19 stacks below the 5′-neighbor C20. In the 3′-direction, stacking between Tg6 and the G7·C18 base pair is disrupted. The solvent-accessible surface area of the Tg nucleotide increases as compared to the native Watson−Crick hydrogen-bonded T·A base pair. An increase in T2 relaxation rates for the Tg6 base protons is attributed to puckering of the Tg base, accompanied by increased disorder at the Tg·G mismatch pair. The axial vs equatorial conformation of the Tg6 CH3 group cannot be determined with certainty from the NMR data. The rMD trajectories suggest that in either the axial or equatorial conformations the cis-5R,6S Tg lesion does not form strong intrastrand hydrogen bonds with the imidazole N7 atom of the 3′-neighbor purine G7. The wobble pairing and disorder of the Tg·G mismatch correlate with the reduced thermodynamic stability of the mismatch and likely modulate its recognition by DNA base excision repair systems.