Base Dynamics in the Hha I Protein Binding Site.

Protein-DNA interactions play an important role in numerous biological functions within the living cell. In many of these interactions, the DNA helix is significantly distorted upon protein-DNA complex formation. The Hha I restriction-modification system is one such system, where the methylation target is flipped out of the helix when bound to the methyltransferase. However, the base flipping mechanism is not well understood. The dynamics of the binding site of the Hha I methyltransferase and endonuclease (underlined) within the DNA oligomer [d(G 1 A 2 T 3 A 4 G 5 C 6 G 7 C 8 T 9 A 10 T 11 C 12 )] 2 are studied using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs deuterated on the base of nucleotides within and flanking the [5'-GCGC-3'] 2 sequence indicate that all of these positions are structurally flexible. Previously, conformational flexibility within the phosphodiester backbone and furanose ring within the target sequence has been observed and hypothesized to play a role in the distortion mechanism. However, whether that distortion was occurring through an active or passive mechanism remained unclear. These NMR data demonstrate that although the [5'-GCGC-3'] 2 sequence is dynamic, the target cytosine is not passively flipping out of the double-helix on the millisecond-picosecond time scale. Additionally, although previous studies have shown that both the furanose ring and phosphodiester backbone experience a change in dynamics upon methylation, which may play a role in recognition and cleavage by the endonuclease, our observations here indicate that methylation has no effect on the dynamics of the base itself.