Effect of adenine methylation on the structure and dynamics of TpA steps in DNA: NMR structure determination of [d(CGAGGTTTAAACCTCG)]2 and its A9-methylated derivative at 750 MHz.

At TpA steps in DNA, the adenine base experiences exceptionally large amplitude (20 degrees-50 degrees) and slow (10 ms-1 microsecond) motion [Kennedy et al. (1993) Biochemistry 32, 8022-8035] which has been correlated with transitions between multiple conformational states [Lefevre et al. (1985) FEBS Lett. 190, 37-40]. The base dynamics can be detected in one-dimensional 1H NMR spectra as excess line width of the aromatic proton resonances. The magnitude of the excess line width is temperature dependent and reaches a maximum at some temperature. In order to better understand the origin of the dynamics, we have studied the effect of N6-methylation of the TpA adenine on both the line widths and its local structure. Here, solution-state 500 and 750 MHz 1H NMR data collected on [d(CGAGGTTTAAACCTCG)]2 show that the excess line width of the TpA adenine-H2 is diminished when the TpA adenine is N6-methylated and that the line width no longer experiences a maximum as the temperature is varied. The resonances sharpen upon methylation because the amplitude of base motion is restricted due to steric effects and due to other structural changes at the TpA site. Additionally, both the TpA adenine-H8 and the exchangeable imino resonance of thymine at the TpA step were also found to have excess line width that is diminished upon N6-methylation. In order to elucidate the structural features responsible for TpA base dynamics, solution-state NMR structures of [d(CGAGGTTTAAACCTCG)]2 and its A9 N6-methylated derivative were determined at 750 MHz. Comparison of the structures shows that poor base stacking at the TpA step may contribute to, or be the origin of, its base dynamics.