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 mu s) motion [Kenn edy et al. (1993) Biochemistry 32, 8022-8035] which has been correlate d with transitions between multiple conformational states [Lefevre et al. (1985) FEES Lett. 190, 37-40]. The base dynamics can be detected i n one-dimensional H-1 NMR spectra as excess line width of the aromatic proton resonances. The magnitude of the excess line width is temperat ure dependent and reaches a maximum at some temperature. In order to b etter understand the origin of the dynamics, we have studied the effec t of N-6-methylation of the TpA adenine on both the line widths and it s local structure. Here, solution-state 500 and 750 MHz H-1 NMR data c ollected on [d(CGAGGTTTAAACCTCG)](2) show that the excess line width o f the TpA adenine-H2 is diminished when the TpA adenine is N-6-methyla ted and that the line width no longer experiences a maximum as the tem perature is varied. The resonances sharpen upon methylation because th e amplitude of base motion is restricted due to steric effects and due to other structural changes at the TpA site. Additionally, both the T pA 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 N-6-methylation. In order to elucidate the structural features re sponsible for TpA base dynamics, solution-state NMR structures of [d(C GAGGTTTAAACCTCG)](2) and its A9 N-6-methylated derivative were determi ned at 750 MHz. Comparison of the structures shows that poor base stac king at the TpA step may contribute to, or be the origin of, its base dynamics.