Base Dynamics in the Hha I Protein Binding Site

Abstract

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 HhaI 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 HhaI methyltransferase and endonuclease (underlined) within the DNA oligomer [d(G₁A₂T₃A₄G₅C₆G₇C₈T₉A₁₀T₁₁C₁₂)]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.

Figure 1

(A) HhaI target DNA, shown in red, showing the flipped deoxycytidine observed in the protein–DNA complex (protein removed). (B) Sequence of the target DNA; red highlighting indicates residues labeled with ²H on the base within and flanking the recognition sequence.

Figure 2

Specific labeling of the bases of A) 2′-deoxyadenosine, B) 2′-deoxyguanosine, C) 2′-thymidine, and D) 2′-deoxycytidine, where the 5-methyl-cytosine replaced the ²H atom at the 5 position of the cytosine ring with an unlabeled methyl group.

Figure 3

Line shape and relaxation simulations of the bases include three independent motions. (A) Slow (rate of 10⁴ Hz) uniform rotation of the DNA helix occurs about the helical symmetry axis, labeled Z. Local motion of the base is referenced to a local coordinate system, where the z axis is indicated by the vector Z_local defined by the angle Θ. The faster (rate 10⁶–10⁹ Hz) local motions of the base are represented by (B) a breathing motion shown by arclike trajectories and (C) small base librations for the bases of each nucleotide.

Figure 4

Seven deuterium line shapes (black) for each of the labeled sites in the nonmethylated and methylated DNA dodecamer with the simulation (blue) of each overlaid. The intensity of the simulated spectra was scaled so that the maximum height of the horns matched that of the experimental spectra. Simulation parameters are described in Table 3.