Biomedical subjects
Publications and source records attributed to L McFail-Isom.
Recent X-ray diffraction, NMR spectroscopy and molecular mechanics results suggest that monovalent cations selectively partition into the minor groove of AT-tracts in DNA. These observations are consistent with DNA deformation by electrostatic collapse around areas of uneven cation density. This model predicts the occurrence of known DNA deformations, such as AT-tract bending and changes in the minor-groove width.
Nucleic acid structure, stability, and reactivity are governed substantially by cations. We propose that magnesium and other biological inorganic ions unstack bases of DNA and RNA. This unstacking function of cations opposes their previously accepted role in stabilizing DNA and RNA duplexes and higher assemblies. We show that cations interact favorably with pi-systems of nucleic acid bases. These cation-pi interactions require access of cations or their first hydration shells to faces of nucleic acid bases. We observe that hydrated magnesium ions located in the major groove of B-DNA pull cytosine bases partially out from the helical stack, exposing pi-systems to positive charge. A series of critical cation-pi interactions contribute to the stability of the anticodon arm of yeast-tRNAphe, and to the magnesium core of the Tetrahymena group I intron P4-P6 domain. The structural consequences of divalent cation-pi interactions are clearly distinct from, and some cases in opposition to, cation-electron lone pair interactions. These observations of cation-pi interactions suggest a number of new mechanistic roles for cations in DNA bending, DNA-protein recognition, base-flipping, RNA folding, and catalysis.
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.
We describe a very accurate addition (called structure X here) to the B-DNA dodecamer family of X-ray structures. Our results confirm the observation of Drew and Dickerson [(1981) J. Mol. Biol. 151, 535-556] that the spine of hydration in AT tract DNA is two layers deep. However, our results suggest that the primary spine is partially occupied by sodium ions. We suggest that many sequence-dependent features of DNA conformation are mediated by site specific binding of cations. For example, preferential localization of cations, as described here within the minor groove of structure X, is probably the structural origin of AT tract bending and groove narrowing. The secondary spine, which does not interact directly with the DNA, is as geometrically regular as the primary spine, providing a model for transmission of sequence information into solvent regions. A fully hydrated magnesium ion located in the major groove of structure X appears to pull cytosine bases partially out from the helical stack, exposing pi-systems to partial positive charges of the magnesium ion and its outer sphere. A partially ordered spermine molecule is located within the major groove of structure X. Dodecamer structures are derived from crystals of [d(CGCGAATTCGCG)]2 in space group P212121 (a = 25 A, b = 40 A, and c = 66 A). On average, those crystals diffracted to around 2.5 A resolution with 2500 unique reflections. Structure X, with the same space group, DNA sequence, and crystal form as the "Dickerson dodecamer", is refined against a complete, low-temperature, 1.4 A resolution data set, with over 11000 reflections. Structure X appears to be conformationally more ordered than previous structures, suggesting that at least a portion of the conformational heterogeneity previously attributed to DNA sequence in fact arises from experimental error.
UV absorbance spectroscopy is the most common method for detecting nucleic acid structural transitions and obtaining thermodynamic parameters. UV-detected melting has been used to determine stabilities of nucleic acid hairpins, duplexes, triplexes, and higher order structures and to determine thermodynamic effects of unusual or modified bases and mismatched base-pairs. We report that in some cases UV absorbance spectroscopy is an inadequate analytical technique for these purposes. Some critical transitions are invisible to UV absorbance spectroscopy. For example, the conversion of dodecamer d(CGCAAATTCGCG) from hairpin to random coil is not accompanied by hyperchromism. Circular dichroism (CD) spectroscopy (263 nm) clearly detects two transitions for this dodecamer, each giving a pronounced change in ellipiticity. The concentration dependence of the low-temperature transition and the concentration independence of the high-temperature transition indicate that the predominant state converts from duplex to hairpin to random coil as the temperature increases. These assignments are confirmed by comparison to oligonucleotides of similar sequence that undergo a hairpin to coil transition only. In contrast to CD spectroscopy, UV absorbance spectroscopy shows only a single transition. The transition detected by UV absorbance spectroscopy corresponds to the low-temperature transition detected by CD. UV absorbance spectroscopy does not detect the second transition at any wavelength (from 218 to 310 nm) (by changes) in either absorbance or its derivative with temperature.
Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.