Narrow Minor Groove Research Articles

Domain architecture of a high mobility group A-type bacterial transcriptional factor.

Myxococcus xanthus transcriptional factor CarD participates in carotenogenesis and fruiting body formation. It is the only reported prokaryotic protein having adjacent "AT-hook" DNA-binding and acidic regions characteristic of eukaryotic high mobility group A (HMGA) proteins. The latter are small, unstructured, nonhistone nuclear proteins that function as architectural factors to remodel DNA and chromatin structure and modulate various DNA binding activities. We find CarD to be predominantly dimeric with two stable domains: (a) an N-terminal domain of defined secondary and tertiary structure which is absent in eukaryotic HMGA proteins; (b) a C-terminal domain formed by the acidic and AT-hook segments and lacking defined structure. CarD, like HMGA proteins, binds specifically to the minor-groove of AT-rich DNA present in two appropriately spaced tracts. As in HMGA proteins, casein kinase II can phosphorylate the CarD acidic region, and this dramatically decreases the DNA binding affinity of CarD. The acidic region, in addition to modulating DNA binding, confers structural stability to CarD. We discuss how the structural and functional plasticity arising from domain organization in CarD could be linked to its role as a general transcriptional factor in M. xanthus.

Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Structural analysis of the binding modes of minor groove ligands comprised of disubstituted benzenes.

Two-dimensional homonuclear NMR was used to characterize synthetic DNA minor groove-binding ligands in complexes with oligonucleotides containing three different A-T binding sites. The three ligands studied have a C(2) axis of symmetry and have the same general structural motif of a central para-substituted benzene ring flanked by two meta-substituted rings, giving the molecules a crescent shape. As with other ligands of this shape, specificity seems to arise from a tight fit in the narrow minor groove of the preferred A-T-rich sequences. We found that these ligands slide between binding subsites, behavior attributed to the fact that all of the amide protons in the ligand backbone cannot hydrogen bond to the minor groove simultaneously.

Preferential counterion binding to A-tract DNA oligomers

The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Tris-acetate buffer, to test the hypothesis that site-specific binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A 3T 3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A 5 tracts or phased runs of T 3A 3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1–20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the A n T n sequence motif.

BII Nucleotides in the B and C Forms of Natural-sequence Polymeric DNA: A New Model for the C Form of DNA

A combination of solid-state 31P and 13C NMR, X-ray diffraction, and model building is used to show that the B and C forms of fibrous macromolecular DNA consist of two distinct nucleotide conformations, which correspond closely to the BI and BII nucleotide conformations known from oligonucleotide crystals. The proportion of the BII conformation is higher in the C form than in the B form. We show structural models for a 10 1 double helix involving BI nucleotides and a 9 1 double helix involving BII nucleotides. The 10 1 BI model is similar to a previous model of B-form DNA, while the 9 1 BII model is novel. The BII model has a very deep and narrow minor groove, a shallow and wide major groove, and highly inclined bases. This work shows that the B to C transition in fibers corresponds to BI to BII conformational changes of the individual nucleotides.

Cations Mediate B-DNA Conformational Heterogeneity

We demonstrate that DNA conformation is sensitive to cationic environment. We describe a high resolution (1.2 A) potassium form of CGCGAATTCGCG, determined from crystals grown in the presence of spermine and magnesium, along with potassium. The structure was refined with anisotropic displacement- factors 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.

Flexible Structure of DNA: Ion Dependence of Minor-Groove Structure and Dynamics

The structure and dynamics of the grooves of DNA are of immense importance for recognition of DNA by proteins and small molecules as well as for the packaging of DNA into nucleosomes and viral particles. Although there is general agreement that the minor groove of DNA varies in a sequence-dependent manner and is narrow in AT regions, alternative models have been presented to explain the molecular basis for the groove narrowing. In one model the groove narrowing results from direct, short-range interactions among DNA bases. In this model the minor groove width of a given sequence is fixed, and any localization of monovalent cations in the groove does not affect the groove structure. In an alternative model the narrow minor groove of A-tracts is proposed to originate from sequence-dependent localization of water and cations. Ion dynamics and exchange make experimental tests of these models difficult, but they can be directly tested by determining how DNA minor-groove structure responds to cation positions i...

Structure and function of the HMGI(Y) family of architectural transcription factors.

The three known members of the HMGI(Y) family of high-mobility group (HMG) mammalian nonhistone nuclear proteins (HMG-I, HMG-Y, and HMGI-C) are thought to participate in numerous biological processes (transcription, replication, retroviral integration, genetic recombination, etc.) by virtue of their ability to recognize and alter the structure of both DNA and chromatin substrates. In vitro and in vivo the HMGI(Y) proteins preferentially bind to the narrow minor groove of stretches of AT-rich DNA by means of highly conserved peptide motifs called AT hooks. In vitro the HMGI(Y) proteins also have the ability to selectively bind to distorted DNA structures and to bend, unwind, and supercoil DNA substrates. Additionally, the HMGI(Y) proteins have the ability to interact with various protein transcription factors both in vitro and in vivo. Specific protein-DNA and protein-protein interactions permit the HMGI(Y) proteins to function as architectural transcription factors that regulate gene expression in vivo by controlling the formation of stereospecific multiprotein complexes on the AT-rich regions of certain gene promoters. Transcriptional overexpression of the HMGI(Y) genes is highly correlated with both cancerous transformation and increased metastatic potential of a number of different cancers, and chromosomal rearrangements involving AT-hook motifs have been associated with various types of benign human mesenchymal tumors. The levels of HMGI(Y) proteins in human cells have been proposed to be sensitive diagnostic indicators of both neoplastic transformation and metastatic progression. Drugs based on the AT-hook motif offer the potential for development of new tumor therapeutic reagents.

Modeling of DNA local parameters predicts encrypted architectural motifs in Xenopus laevis ribosomal gene promoter.

We have modeled local DNA sequence parameters to search for DNA architectural motifs involved in transcription regulation and promotion within the Xenopus laevis ribosomal gene promoter and the intergenic spacer (IGS) sequences. The IGS was found to be shaped into distinct topological domains. First, intrinsic bends split the IGS into domains of common but different helical features. Local parameters at inter-domain junctions exhibit a high variability with respect to intrinsic curvature, bendability and thermal stability. Secondly, the repeated sequence blocks of the IGS exhibit right-handed supercoiled structures which could be related to their enhancer properties. Thirdly, the gene promoter presents both inherent curvature and minor groove narrowing which may be viewed as motifs of a structural code for protein recognition and binding. Such pre-existing deformations could simply be remodeled during the binding of the transcription complex. Alternatively, these deformations could pre-shape the promoter in such a way that further remodeling is facilitated. Mutations shown to abolish promoter curvature as well as intrinsic minor groove narrowing, in a variant which maintained full transcriptional activity, bring circumstantial evidence for structurally-preorganized motifs in relation to transcription regulation and promotion. Using well documented X. laevis rDNA regulatory sequences we showed that computer modeling may be of invaluable assistance in assessing encrypted architectural motifs. The evidence of these DNA topological motifs with respect to the concept of structural code is discussed.

A-tract bending: insights into experimental structures by computational models

While solution structures of adenine tract (A-tract) oligomers have indicated a unique bend direction equivalent to negative global roll (commonly termed “minor-groove bending”), crystallographic data have not unambiguously characterized the bend direction; nevertheless, many features are shared by all A-tract crystal and solution structures (e.g. propeller twisting, narrow minor grooves, and localized water spines). To examine the origin of bending and to relate findings to the crystallographic and solution data, we analyze molecular dynamics trajectories of two solvated A-tract dodecamers: 1D89, d(CGCGA6CG), and 1D98, d(CGCA6GCG), using a new general global bending framework for analyzing bent DNA and DNA/protein complexes. It is significant that the crystallographically-based initial structures are converted from dissimilar to similar bend directions equivalent to negative global roll, with the average helical-axis bend ranging from 10.5 ° to 14.1 °. The largest bend occurs as positive roll of 12 ° on the 5′ side of the A-tracts (supporting a junction model) and is reinforced by gradual curvature at each A-tract base-pair (bp) step (supporting a wedge model). The precise magnitude of the bend is subtly sequence dependent (consistent with a curved general sequence model). The conversion to negative global roll only requires small local changes at each bp, accumulated over flexible moieties both outside and inside the A-tract. In contrast, the control sequence 1BNA, d(CGCGA2TTCGCG), bends marginally (only 6.9 °) with no preferred direction. The molecular features that stabilize the bend direction in the A-tract dodecamers include propeller twisting of AT base-pairs, puckering differences between A and T deoxyriboses, a narrow minor groove, and a stable water spine (that extends slightly beyond the A-tract, with lifetimes approaching 0.2 ns). The sugar conformations, in particular, are proposed as important factors that support bent DNA. It is significant that all these curvature-stabilizing features are also observed in the crystallographic structures, but yield overall different bending paths, largely due to the effects of sequences outside the A-tract. These results merge structural details reported for A-tract structures by experiment and theory and lead to structural and dynamic insights into sequence-dependent DNA flexibility, as highlighted by the effect of an A-tract variant of a TATA-box element on bending and flexibility required for TBP binding.

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes.

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)](2), in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)](2)as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)](2). Long-lived water molecules with correlation time tau(c)longer than 0.3 ns were found close to the RNA adenine H2 and H1' protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA-RNA junction, adopt C1'-exo or C2'-endo conformations. The exchange rates for RNA C2'-OH were found to be approximately 5-20 s(-1). This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2), which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2)is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA. RNA hybrid and chimeric duplexes.

Hydration of [d(CGC)r(aaa)d(TTTGCG)] 2

We have studied the hydration and dynamics of RNA C2′-OH in a DNA·RNA hybrid chimeric duplex [d(CGC)r(aaa)d(TTTGCG)] 2. Long-lived water molecules with correlation time τ c larger than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not to the other two thymine bases (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4′- endo sugar conformation (intermediate between B-form and A-form), while the other DNA residues including 3C in the DNA-RNA junction, adopt C1′- exo or C2′- endo conformations (in the B-form domain). Based on the NOE cross-peak patterns, we have found that RNA C2′-OH tends to orient toward the O3′ direction, forming a possible hydrogen bond with the 3′-phosphate group. The exchange rates for RNA C2′-OH were found to be around 5–20 s −1, compared to 26.7(±13.8) s −1 reported previously for the other DNA·RNA hybrid duplex. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)] 2, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The distinct hydration patterns of the RNA adenine H2 and H1′ protons and the DNA 7T methyl group in the hybrid segment, as well as the orientation and dynamics of the RNA C2′-OH protons, may provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.

Sequence-dependent variation in DNA minor groove width dictates orientational preference of Hoechst 33258 in A-tract recognition: solution NMR structure of the 2:1 complex with d(CTTTTGCAAAAG)(2).

The solution structure of the dodecamer duplex d(CTTTTGCAAAAG)(2)and its 2:1 complex with the bis -benzimidazole Hoechst 33258 has been investigated by NMR and NOE-restrained molecular dynamics (rMD) simulations. Drug molecules are bound in each of the two A-tracts with the bulky N-methylpiperazine ring of each drug located close to the central TG (CA) step, binding essentially to the narrow minor groove of each A-tract. MD simulations over 1 ns, using an explicit solvation model, reveal time-averaged sequence-dependent narrowing of the minor groove from the 3'-end towards the 5'-end of each TTTT sequence. Distinct junctions at the TpG (CpA) steps, characterised by large positive roll, low helical and propeller twists and rapid AT base pair opening rates, add to the widening of the groove at these sites and appear to account for the bound orientation of the two drug molecules with the N-methylpiperazine ring binding in the wider part of the groove close to the junctions. Comparisons between the free DNA structure and the 2:1 complex (heavy atom RMSD 1.55 A) reveal that these sequence-dependent features persist in both structures. NMR studies of the sequence d(GAAAAGCTTTTC)(2), in which the A-tracts have been inverted with the elimination of the TpG junctions, results in loss of orientational specificity of Hoechst 33258 and formation of multiple bound species in solution, consistent with the drug binding in a number of different orientations.

C-H..O hydrogen bonds in minor groove of A-tracts in DNA double helices

Analysis of available B-DNA type oligomeric crystal structures as well as protein-bound DNA fragments (solved using data with resolution <2.6 Å) indicates that in both data sets, a majority of the (3′-Ade) H2..O2(3′-Thy/Cyt) distances in AA.TT and GA.TC dinucleotide steps, are considerably shorter than their values in a uniform fibre model, and are smaller than their optimum separation distance. Since the electropositive C2-H2 group of adenine is in close proximity of the electronegative keto oxygen atoms of both pyrimidine bases in the antiparallel strand of the double-helical DNA structures, it suggests the possibility of intra-base-pair as well as cross-strand C-H..O hydrogen bonds in the minor groove. The C2-H2..O2 hydrogen bonds within the A.T base-pairs could be a natural consequence of Watson-Crick pairing. However, the close cross-strand interactions between the bases at the 3′-ends of the AA.TT and GA.TC steps arise due to the local sequence-dependent geometry of these steps. While the base-pair propeller twist in these steps is comparable to the fibre model, some of the other local parameters such as base-pair opening angle and inter-base-pair slide show coordinated changes, leading to these shorter C2-H2..O2 distances. Hence, in addition to the well-known minor groove hydration, it appears that favourable C2-H2..O2 cross-strand interactions may play a role in imparting a characteristic geometry to AA.TT and GA.TC steps, as well as A n . T n and GA n . T n C tracts, which leads to a narrow minor groove in these regions.

Binding of the Delta- and Lambda-Enantiomers of [Ru(dmphen)(2)dpq](2+) to the Hexanucleotide d(GTCGAC)(2).

1H NMR spectroscopy and viscosity measurements have been used to study the oligonucleotide binding of the Delta- and Lambda-enantiomers of the metal complex [Ru(dmphen)(2)dpq](2+) (dmphen = 2,9-dimethyl-1,10-phenanthroline and dpq = dipyrido[3,2-f:2',3'-h]quinoxaline). The addition of either enantiomer to d(GTCGAC)(2) induced large upfield shifts and significant broadening for the hexanucleotide imino and metal complex dpq resonances. These data coupled with the observed increase in the melting transition midpoint of the hexanucleotide duplex upon addition of either enantiomer suggests that both Delta- and Lambda-[Ru(dmphen)(2)dpq](2+) bind by intercalation. A significant number of metal complex to hexanucleotide NOE contacts were observed in NOESY spectra of d(GTCGAC)(2) with added Delta- or Lambda-[Ru(dmphen)(2)dpq](2+). The observed intermolecular NOEs were consistent with both enantiomers intercalating between the G(4)A(5) bases of one strand and the T(2)C(3) bases of the complementary strand. Intermolecular NOEs from the dmphen protons were only observed to protons located in the hexanucleotide minor groove. Alternatively, NOE contacts from the dpq protons were observed to both major and minor groove protons. The NOE data suggest that the dpq ligand of the Delta-enantiomer intercalates deeply into the hexanucleotide base stack while the Lambda-enantiomer can only partially intercalate. Viscosity measurements were consistent with the proposed intercalation binding models. The addition of the Delta-enantiomer increased the relative viscosity of the DNA solution, while a decrease in the relative viscosity of the DNA was observed upon addition of the Lambda-metal complex. These results confirm our proposal that octahedral metallointercalators can intercalate from the minor groove. In addition, the results demonstrate that the left-handed enantiomer of [Ru(dmphen)(2)dpq](2+) prefers to intercalate from the narrow minor groove despite only being able to partially insert a polycyclic aromatic ligand into the DNA base stack.

Solution structure and dynamics of the A-T tract DNA decamer duplex d(GGTAATTACC)2: implications for recognition by minor groove binding drugs

The structure of the DNA decamer duplex d(GGTAATTACC)(2) has been determined using NMR distance restraints and molecular dynamics simulations of 500 ps to 1 ns in aqueous solution at 300 K. Using both canonical A and canonical B starting structures [root-mean-square deviation (RMSD) 4.6 A; 1 A=10(-10) m], with and without experimental restraints, we show that all four simulations converge to a similar envelope of final conformations with B-like helical parameters (pairwise RMSD 1.27-2.03 A between time-averaged structures). While the two restrained simulations reach a stable trajectory after 300-400 ps, the unrestrained trajectories take longer to equilibrate. We have analysed the dynamic aspects of these structures (sugar pucker, helical twist, roll, propeller twist and groove width) and show that the minor groove width in the AATT core of the duplex fluctuates significantly, sampling both wide and narrow conformations. The structure does not have the highly pre-organized narrow minor groove generally regarded as essential for recognition and binding by small molecules, suggesting that ligand binding carries with it a significant component of 'induced-fit'. Our simulations show that there are significant differences in structure between the TpA step (where p=phosphate) and the ApA and ApT steps, where a large roll into the major groove at the TpA step appears to be an important factor in widening the minor groove at this position.

Heterogeneity in the actions of drugs that bind in the DNA minor groove.

Distamycin and Hoechst 33258 have long served as the model compounds for biochemical, biophysical, and clinical studies of the drugs that bind in the DNA minor groove. However, the results presented in this investigation clearly show that 4,6-diamidino-2 phenylindole (DAPI) is superior to both of these drugs at negating the effects of intrinsic DNA curvature and anisotropic bendability as measured by electrophoretic and ligation analysis. In addition, DAPI was more effective than distamycin and Hoechst 33258 at inhibiting the assembly of nucleosomes onto synthetic and natural sequences that have multiple closely spaced oligo-AT sequences that serve as drug binding sites. Since these effects may be related to the biological action of the drugs, it was of interest to determine the mechanism that was responsible for the enhanced action of DAPI. The possibility that the differential drug potencies resulted from differential overall affinities of the ligands for A-tract molecules was considered, but drug binding studies suggested that this was not the case. It is also unlikely that the differential drug effects resulted from the binding of the drugs to different DNA sites since the oligo A/T binding sites for DAPI and Hoechst were centered on the same nucleotide positions as revealed by footprinting studies using exonuclease III, DNase I, and hydroxyl radical. However, the footprinting studies with DNase I did uncover a potentially important difference between the drugs. DAPI protected only the AT bp in the binding sites, while distamycin and Hoechst protected these bp as well as flanking Gs and Cs. These results permitted us to advance a preliminary model for the enhanced action DAPI. According to the model, the short length of DAPI and its absolute specificity for A/T bps with narrow minor grooves ensures that only particularly minor grooves that give rise to curvature and anisotropic bendability are occupied by the drug. Consequently, each helical deflection induced by an A-tract in the absence of the drug is countered by an opposite deflection induced by DAPI binding, thus effectively neutralizing intrinsic curvature and bending into the minor groove.

DNA minor groove recognition of a non-self-complementary AT-rich sequence by a tris-benzimidazole ligand.

The crystal structure of the non-self-complementary dodecamer DNA duplex formed by d(CG[5BrC]ATAT-TTGCG) and d(CGCAAATATGCG) has been solved to 2.3 A resolution, together with that of its complex with the tris-benzimidazole minor groove binding ligand TRIBIZ. The inclusion of a bromine atom on one strand in each structure enabled the possibility of disorder to be discounted. The native structure has an exceptional narrow minor groove, of 2.5-2.6 A in the central part of the A/T region, which is increased in width by approximately 0.8 A on drug binding. The ligand molecule binds in the central part of the sequence. The benzimidazole subunits of the ligand participate in six bifurcated hydrogen bonds with A:T base pair edges, three to each DNA strand. The presence of a pair of C-H...O hydrogen bonds has been deduced from the close proximity of the pyrrolidine group of the ligand to the TpA step in the sequence.

An NMR Study of d(CTACTGCTTTAG).d(CTAAAGCAGTAG) Showing Hydration Water Molecules in the Minor Groove of a TpA Step

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-Hl′(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment ∼ 3.9Å) and a sudden widening at the T10:A15 base-pair (∼ 5.3Å), the standard B-DNA distance being ∼ 5Å. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n > 2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for Al 1 ending the 5′TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the Al 1:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to Al 1 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.

Structure of the potassium form of CGCGAATTCGCG: DNA deformation by electrostatic collapse around inorganic cations.

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.