Structure of the B-DNA decamer C-C-A-A-C-I-T-T-G-G in two different space groups: conformational flexibility of B-DNA.

For the first time, the same B-DNA oligomer has been crystallized and its structure solved in two different space groups. Crystallization of C-C-A-A-C-I-T-T-G-G with Ca2+ yields monoclinic space group C2 with a = 31.87 A, b = 25.69 A, c = 34.21 A, beta = 114.1 degrees, and five base pairs per asymmetric unit. The 5026 2 sigma data to 1.3 A refine to R = 0.152 with 72 waters, one heptavalent hydrated calcium complex, and one cacodylate ion per asymmetric unit. In contrast, crystallization with Mg2+ yields trigonal space group P3(2)21 with a = b = 33.23 A, c = 94.77 A, gamma = 120 degrees, and 10 base pairs per asymmetric unit. The 1725 2 sigma data to 2.2 A refine to R = 0.164 with 36 water molecules and one octahedral magnesium complex per asymmetric unit. The monoclinic form is virtually isostructural with previously solved monoclinic decamers, including twist angles of ca. 50 degrees at C-A and T-G steps. In contrast, the trigonal structure has quite different local helix parameters, with twist angles of ca. 36 degrees at the corresponding steps. These local parameter differences can only be attributed to crystal packing, suggesting that certain sequences of B-DNA are more flexible and influenced by their surroundings than had previously been thought. Such deformability may be important for interaction of B-DNA with control proteins, where both static structure and dynamic deformability comprise components of the recognition process. The crossing of two helices at an angle of 120 degrees in the trigonal cell is a model for an antiparallel, uncrossed Holliday junction, as has been noted earlier by Timsit and Moras [Timsit, Y., & Moras, D. (1991) J. Mol. Biol. 221, 919-940] from a rhombohedral DNA dodecamer structure analysis.

Patterson methods in fibre diffraction analysis.

The potential of the Patterson methods for X-ray diffraction studies of textures is examined in DNA fibres. Patterson analysis, which is rarely used in these situations, is shown to yield important information on the preliminary interpretation of diffraction patterns and to increase the reliability of the three-dimensional structural pattern obtained for polymeric molecules. We also show how the screw symmetry of helical molecules can be used to calculate their electron density by means of the three-dimensional Patterson function.

Sequence effects on local DNA topology.

Nuclear Overhauser effect-derived distances between adenine H2 protons and anomeric H1' protons on the same strand or on the complementary strand are presented for several different DNA duplexes. The cross-strand (n)AH2 to (m + 1)H1' distances [designated as x, where (n) and (m) are complementary residues] vary by up to 1 A depending on the sequence. In all possible A-containing pyrimidine-purine steps (CA, TG, and TA), x is greater than 4.5 A. In GA steps, x varies within rather wide limits in the range 3.8-4.5 A, whereas in AA steps the lower limit is 3.7 A and the upper limit is approximately 4.2 A. In purine-purine steps, x is affected by at least three factors: (i) adjacent pyrimidine-purine steps at the 5' end [e.g., YRA sequences (where Y = T or C and R = G or A)], or a pyrimidine-purine step at the 3' end of the pyrimidine-pyrimidine step on the complementary strand, cause x to increase, (ii) an AT step at the 3' end of a purine-purine step (e.g., RAT) causes x to decrease, and (iii) substitution of bases at the next-nearest neighbor position leads to changes in x at GA and AA steps. The latter factor seems to be due to a cooperative effect arising from formation of the "anomalous" B' structure when the substitution produces an AnTm tract (which always produces a decrease in x). The data indicate that (n)AH2-(n + 1)H1' distances on the same strand (designated as s) are also sequence dependent. Thus on AA steps, neighboring substitutions produce the same effect on s as on the cross-strand x distances. The results lead to the ability to predict changes in AH2-H1' distances depending on the DNA sequence. By using high-resolution x-ray B-type structures as a set of allowable B conformations, a very good correlation was found between x and the minor groove width parameters P-P or H1'-H1'. Thus, the x distances are a direct probe of the minor groove width in B-type DNA, and changes in this distance therefore reflect changes in the minor groove width. Since many of the sequences studied are sites of protein recognition, the observed sequence-structure dependence in DNA probably plays an important role in the process of recognition by proteins and minor groove ligands such as drugs.

Low-temperature crystallographic analyses of the binding of Hoechst 33258 to the double-helical DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G.

The crystal structure of the complex of Hoechst 33258 and the DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G has been solved from X-ray data collected at three different low temperatures (0, -25, and -100 degrees C). Such temperatures have permitted collection of higher resolution data (2.0, 1.9, and 2.0 A, respectively) than with previous X-ray studies of the same complex. In all three cases, the drug is located in the narrow central A-A-T-T region of the minor groove. Data analyses at -25 and -100 degrees C (each with a 1:1 drug/DNA ratio in the crystallizing solution) suggest a unique orientation for the drug. In contrast, two orientations of the drug were found equally possible at 0 degrees C with a 2:1 drug/DNA ratio in solution. Dihedral angles between the rings of Hoechst 33258 appear to change in a temperature-dependent manner. The drug/DNA complex is stabilized by single or bifurcated hydrogen bonds between the two N-H hydrogen-bond donors in the benzimidazole rings of Hoechst and adenine N3 and thymine O2 acceptors in the minor groove. A general preference for AT regions is conferred by electrostatic potential and by narrowing of the walls of the groove. Local point-by-point AT specificity follows from close van der Waals contacts between ring hydrogen atoms in Hoechst 33258 and the C2 hydrogens of adenines. Replacement of one benzimidazole ring by purine in a longer chain analogue of Hoechst 33258 could make that particular site GC tolerant in the manner observed at imidazole substitution for pyrrole in lexitropsins.

Charge grouping approaches to calculation of electrostatic forces in molecular dynamics of macromolecules.

Calculation of long-range electrostatic interactions is the most time-consuming step in theoretical simulation of the structure and dynamics of macromolecules. In practice very short cutoff distances are used, which may distort the behavior of the model system. We describe two accurate approaches to calculation of electrostatic forces based on hierarchical grouping of charges into cubes. The first is similar to the O(NlogN) algorithm developed by Barnes, J. and Hut, P., Nature (London) 324, 446-449 (1986), for simulation of a gravitational motion of N bodies. The second approach we formulate for a system with periodic boundary conditions in the nearest image approximation. The calculation of electrostatic interactions and a charge grouping procedure are faster than O(N2). The average inaccuracy in the force introduced by the grouping does not exceed 1%. We describe a small modification of the same approach which makes it suitable for long strongly charged polymers as well. This accurate approach to calculation of electrostatic interactions is illustrated with an example of the dynamics of ions near DNA. Quick equilibration of the ionic distribution is observed during molecular dynamics simulation if electrostatic forces are properly calculated, while the behavior and distribution of ions are less realistic when the conventional cutoff distances are used.

Disordered single crystal evidence for a quadruple helix formed by guanosine 5'-monophosphate.

The disodium salt of guanosine 5'-monophosphate (5'-GMP) has been crystallized earlier in an orthorhombic array. We have obtained a new crystal form of 5'-GMP at pH 8 which reveals a clear helical nature, with guanine bases stacked perpendicular to the helix axis. Although the X-ray pictures show partial disorder, they can be indexed on a hexagonal net with a = b = 28.6 A, c = 9.8 A, V = 6942 A3 (1A = 0.1 nm). The probable space group is P6(4), and past experience with ca. 600 A3 per base in oligonucleotide crystals suggests that the cell contains 12 GMP molecules. The crystal packing parameters and the intensity distribution agree with a model of three hydrogen-bonded guanine tetrads in the unit cell, stacked so as to build a quadruple helix similar to that proposed earlier from fiber studies (Zimmerman, S.B., J. Mol. Biol. 106, 663-672 (1976)).

Bh-DNA: variations of the poly[d(A)].poly[d(T)] structure within the framework of the fibre diffraction studies.

A refinement of the recent results for poly[d(A)].poly[d(T)] (Alexeev et al., J. Biomol. Struct. Dyn. 4,989 (1987)) involving additional parameters of the base-pair structure and of the sugar-phosphate backbone expands the conformational potential of this polynucleotide of the B type to include the possibility of bifurcated hydrogen bonds of the kind recently discovered in crystalline deoxyoligonucleotide with lone d(A)n.d(T)n stretch (Nelson et al., Nature 330, 221 (1987)). Still, analysis of the available data and energy calculations do not seem to indicate that the bifurcated H-bonds are a crucial factor responsible for the anomalous structure of the d(A)n.d(T)n sequence. The unique structural properties of poly[d(A)].poly[d(T)] can hardly be explained without taking into account its interactions with the double-layer hydration spine in the minor groove. In view of the hydration mechanism stabilizing poly[d(A)].poly[d(T)] and of the polynucleotide's heteronomous prehistory (Arnott et al., Nucleic Acids Res. 11,4141 (1983)) we suggest that this B-type structure be called Bh.

G-DNA: a twice-folded DNA structure adopted by single-stranded oligo(dG) and its implications for telomeres.

Our dimethyl sulfate modification experiments suggest that (dG)n stretches within single-stranded DNA fragments, which represent the simplest model for telomeric sequences, adopt a complex intrastrand structure other than a simple hairpin. We present a molecular model for the DNA structure that conforms to dimethyl sulfate methylation data. The principal element of this G-DNA structure is a quadruple helix formed by pairwise antiparallel segments of the twice-folded (dG)n stretch. This quadruple core has two wide and two narrow grooves connected by three loop-shaped segments. The strong stacking interactions of the neighboring guanine tetrads and the large number of hydrogen bonds formed can be the primary reasons that such structures are favored over a common hairpin for long (dG)n stretches. Such compact structures may be formed from (dG)n stretches of telomeric sequences.

DNA B to D transition can be explained in terms of hydration economy of the minor groove atoms.

Adjacent phosphate oxygen atoms in A and Z-DNA are located much closer together than in the B form and can be hydrated more economically due to the formation of water bridges between them, whereas in the B form phosphates are hydrated individually. This principle of hydration economy of phosphate groups discovered by Saenger and colleagues could not be applied to the B-D transition, which, like the B-A and B-Z transitions, occurs in a situation of water deficiency, because the distances between adjacent phosphates of individual polynucleotide chains in the D form are not much different from B-DNA. It follows from our calculations of B and D-DNA accessibility to solvent performed by the method of Lee & Richards, and from a simulation of solvent structure near DNA, that there is an economy of hydration only for the minor groove atoms. This feature and some experimental data can explain why only a limited range of sequences consisting of A.T or I.C pairs undergo the transition to the D form. The conformational transition in DNAs with such sequences to a poly[d(A]).poly[d(T])-like conformation (Bh-DNA), which is accompanied by a narrowing of the minor groove, can be explained in the same way. Calculations suggest that in the D-form minor groove of different A-T or I-C DNAs there is a double-layer hydration spine similar to that observed by Drew & Dickerson in the A-T tract of the d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer. The B-D and B-Bh transitions in A + T-rich DNAs can have biological implications, e.g. they can facilitate DNA bending upon the interaction with proteins.

[Structure of poly(dA).poly(dT) from data of x-ray diffraction, energy calculations and nuclear magnetic resonance]. / Struktura poli(dA).poli(dT) po dannym rentgenovskoi difraktsii, énergeticheskikh rashetov i iadernogo magnitnogo rezonansa.

The results of X-ray diffraction studies of poly(dA).poly(dT) have been compared with the results of energy optimization and with the NMR data in solution. Slight refinement of the X-ray and energetically optimal models leads to a very good quantitative agreement with the NMR data, that suggests similarity of the poly(dA).poly(dT) structure in a condensed state and in solution. One of the features distinguishing these models from the classic B form is a narrowed minor groove of the double helix. The anomalous properties of DNA with this sequence can be related specific organization of the water molecules near the polynucleotide.

The structure of poly(dA):poly(dT) in a condensed state and in solution.

New X-ray and energetically optimal models of poly(dA):poly(dT) with the hydration spine in the minor groove have been compared with the NMR data in solution (Behling, R.W. and Kearns, D.R. (1986) Biochemistry 25, 3335-3346). These models have been refined to achieve a better fit with the NMR data. The obtained results suggest that the poly(dA):poly(dT) structure in a condensed state is similar to that in solution. The proposed conformations of poly(dA):poly(dT), unlike the classic B form, satisfy virtually all geometrical requirements which follow from the NMR data. Thus, the X-ray and energetically optimal poly(dA):poly(dT) structures (or those with slight modifications) can be considered as credible models of the poly(dA):poly(dT) double helix in solution. One of the features distinguishing these models from the classic B form is a narrowed minor groove.

The structure of poly(dA).poly(dT) as revealed by an X-ray fibre diffraction.

X-ray diffraction in fibres revealed that the calcium salt of poly(dA).poly(dT) is a 10-fold double helix with a pitch of 3.23 nm. The opposite sugar-phosphate chains in the refined model are characterized by a complete conformational equivalence and contain sugars in a conformation close to C2'-endo. As a result a new model of the sodium salt of poly(dA).poly(dT) has been constructed, which is different from the Heteronomous DNA proposed earlier (S. Arnott et al., Nucl. Acids Res. 11, 4141 (1983)). The new model of Na-poly(dA).poly(dT) has conformationally similar opposite chains; it is a structure of the B-type, rather like that of Ca-poly(dA).poly(dT).

Poly(dA).poly(dT) is a B-type double helix with a distinctively narrow minor groove.

The structure of poly(dA).poly(dT) currently arouses great interest, mainly because dAn.dTn stretches are associated with considerable DNA bending. Until recently the heteronomous DNA described by Arnott et al., with the poly(dA) and poly(dT) chains in A and B conformations respectively, was the only detailed model of this structure. Following our earlier studies of the interaction of DNA and monovalent ions, we examined the X-ray diffraction of the bivalent Ca2+ salt of poly(dA).poly(dT) (Ca-poly(dA).poly(dT)) and found no sign of a heteronomous structure: Ca-poly(dA).poly(dT) in fibres shows fully equivalent B-type conformations of the opposite sugar-phosphate chains. A revision of the structure of the sodium salt, Na-poly(dA).poly(dT), based on this result, yields only a slightly heteronomous structure with each chain in a B-type conformation, which is in much better agreement with the experimental data underlying the original heteronomous model. Both structures, Ca- and Na-poly(dA).poly(dT), have a minor groove narrower than that of the B form: this peculiarity seems to be very important for the interaction of poly(dA).poly(dT) and biologically significant molecules (including proteins and antibiotics). The specific base-pair positions in poly(dA).poly(dT) may account for the DNA bending adjacent to dAn.dTn tracts.