B-DNA structure is intrinsically polymorphic: even at the level of base pair positions

Abstract

Increasingly exact measurement of single crystal X-ray diffraction data offers detailed characterization of DNA conformation, hydration and electrostatics. However, instead of providing a more clear and unambiguous image of DNA, highly accurate diffraction data reveal polymorphism of the DNA atomic positions and conformation and hydration. Here we describe an accurate X-ray structure of B-DNA, painstakingly fit to a multistate model that contains multiple competing positions of most of the backbone and of entire base pairs. Two of ten base-pairs of CCAGGCCTGG are in multiple states distinguished primarily by differences in slide. Similarly, all the surrounding ions are seen to fractionally occupy discrete competing and overlapping sites. And finally, the vast majority of water molecules show strong evidence of multiple competing sites. Conventional resolution appears to give a false sense of homogeneity in conformation and interactions of DNA. In addition, conventional resolution yields an average structure that is not accurate, in that it is different from any of the multiple discrete structures observed at high resolution. Because base pair positional heterogeneity has not always been incorporated into model-building, even some high and ultrahigh-resolution structures of DNA do not indicate the full extent of conformational polymorphism.

Figure 1.

(A) The structure of CCAGGCCTGG^0.96 showing indicating multiple states. State A is blue and state B is yellow. The bases are numbered 1–10 in the first strand, 11–20 in the other. (B) The 2F_o − F_c electron density map surrounding the DNA only (blue net), contoured at 1σ.

Figure 2.

(A) 2F_o − F_c electron electron density map (sum map) showing a base pair [C(1)–G(11)] that is found in a single position. (B) Electron density showing two discrete positions of the central G(5)–C(16) base pair. This map is unbiased in that phases were calculated from the initial, single-position model even though atoms of both states are shown. (C) Difference electron density surrounding the central G(5)–C(16) base pair. The positive difference density is green, indicating where additional base atoms should added to give a better fit of the model to the data. These difference maps is unbiased, with phases calculated from the initial, single-position model. The atoms of the final multiple-position model are shown but were not used for phase calculation. The negative difference density is red, indicating where atoms should be removed from the model to give a better fit. For all three panels, the sum maps are contoured at contoured at 1σ. Difference maps are contoured at 2.5σ.

Figure 3.

The G(5)–C(16) base pair is in two discrete positions.

Figure 4.

The major groove. Three hexahydrated magnesium complexes complete for overlapping sites within the major groove of the DNA. The data here support a model in which a magnesium ion in solution would shift between sites, with occupancy of only one site at a time. In the X-ray structure, these complexes are partially occupied, indicating occupancy on an either/or basis in the crystalline ensemble.