The potassium form of d(CGCGAATTCGCG) solved by X-ray diffraction to 1.75 A resolution indicates that monovalent cations penetrate the primary and secondary layers of the "spine of hydration". Both the sodium [Shui, X., McFail-Isom, L., Hu, G. G., and Williams, L. D. (1998) Biochemistry 37, 8341-8355] and the potassium forms of the dodecamer at high resolution indicate that the original description of the spine, only two layers deep and with full occupancy by water molecules, requires substantive revision. The spine is merely the bottom two layers of a four layer solvent structure. The four layers combine to form a repeating motif of fused hexagons. The top two solvent layers were not apparent from previous medium-resolution diffraction data. We propose that the narrow minor groove and axial curvature of A-tract DNA arise from localization of cations within the minor groove. In general, the results described here support a model in which most or all forces that drive DNA away from canonical B-conformation are extrinsic and arise from interaction of DNA with its environment. Intrinsic forces, originating from direct base-base interactions such as stacking, hydrogen bonding, and steric repulsion among exocyclic groups appear to be insignificant. The time-averaged positions of the ubiquitous inorganic cations that surround DNA are influenced by DNA bases. The distribution of cations depends on sequence. Regions of high and low cation density are generated spontaneously in the solvent region by heterogeneous sequence or even within the grooves of homopolymers. The regions of high and low cation density deform DNA by electrostatic collapse. Thus, the effects of small inorganic cations on DNA structure are similar to the effects of proteins.

Here we demonstrate that monovalent cations can localize around B-DNA in geometrically regular, sequence-specific sites in oligonucleotide crystals. Positions of monovalent ions were determined from high-resolution X-ray diffraction of DNA crystals grown in the presence of thallium(I) cations (Tl(+)). Tl(+) has previously been shown to be a useful K(+) mimic. Tl(+) positions determined by refinement of model to data are consistent with positions determined using isomorphous F(Tl) - F(K) difference Fouriers and anomalous difference Fouriers. None of the observed Tl(+) sites surrounding CGCGAATTCGCG are fully occupied by Tl(+) ions. The most highly occupied sites, located within the G-tract major groove, have estimated occupancies ranging from 20% to 35%. The occupancies of the minor groove sites are estimated to be around 10%. The Tl(+) positions in general are not in direct proximity to phosphate groups. The A-tract major groove appears devoid of localized cations. The majority of the observed Tl(+) ions interact with a single duplex and so are not engaged in lattice interactions or crystal packing. The locations of the cation sites are dictated by coordination geometry, electronegative potential, avoidance of electropositive amino groups, and cation-pi interactions. It appears that partially dehydrated monovalent cations, hydrated divalent cations, and polyamines compete for a common binding region on the floor of the G-tract major groove.

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We demonstrate that DNA conformation is sensitive to cationic environment. We describe a high resolution (1.2 Å) potassium form of CGCGAATTCGCG, determined from crystals grown in the presence of spermine and magnesium, along with potassium. The structure was refined with anisotropic displacementfactors by SHELX-97 to an R-factor of 13.9%. A comparison of this structure with others, reveals that the conformation of CGCGAATTCGCG varies in direct response to cation type and position. The DNA conformation in the presence of excess magnesium differs from the conformation in the presence of excess spermine. Divalent cations near the minor groove sequester into the lip, which is the region between opposing phosphate groups. Minor groove width is sensitive to, and can be predicted by, cation positions. It appears that minor groove narrowing is facilitated by interactions of cations with opposing phosphate groups.

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