Base Dynamics in the HhaI Protein Binding Site

Protein-DNA interactions play an important rolein numerousbiological functions within the living cell. In many of these interactions,the DNA helix is significantly distorted upon protein-DNA complexformation. The HhaI restriction-modification systemis one such system, where the methylation target is flipped out ofthe helix when bound to the methyltransferase. However, the base flippingmechanism is not well understood. The dynamics of the binding siteof the HhaI 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 nucleotideswithin and flanking the [5 & PRIME;-GCGC-3 & PRIME;](2) sequenceindicate that all of these positions are structurally flexible. Previously,conformational flexibility within the phosphodiester backbone andfuranose ring within the target sequence has been observed and hypothesizedto play a role in the distortion mechanism. However, whether thatdistortion was occurring through an active or passive mechanism remainedunclear. These NMR data demonstrate that although the [5 & PRIME;-GCGC-3 & PRIME;](2) sequence is dynamic, the target cytosine is not passivelyflipping out of the double-helix on the millisecond-picosecondtime scale. Additionally, although previous studies have shown thatboth the furanose ring and phosphodiester backbone experience a changein dynamics upon methylation, which may play a role in recognitionand cleavage by the endonuclease, our observations here indicate thatmethylation has no effect on the dynamics of the base itself.