Molecular structure of a complete turn of A-DNA.

We have determined the crystal structure of the dodecamer d(CCCCCGCGGGGG), showing for the first time a complete turn of A-DNA. It has average structural parameters similar to those determined in fibres. Nevertheless it shows a considerable local variation in structure which is in part associated with the presence of a bound spermine molecule. We conclude that the local DNA conformation does not only depend on the base sequence, but may be strongly modified upon interaction with other molecules. In particular, the CpG sequence, which is found in hypersensitive regions of the genome, appears to be able to easily change its conformation under external influences.

Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution.

Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.

Crystallization and preliminary X-ray analysis of the DNA decamers d(CCGGATCCGG) and d(CCGGCGCCGG).

The DNA decamers d(CCGGATCCGG) and d(CCGGCGCCGG) have been crystallized for X-ray analysis in order to investigate the effects of changing the two central base pairs of the DNA fragment d(CCGGGACCGG). Previous studies have already demonstrated that the structure of the former DNA fragment contains a DNA Holliday junction. Crystals were obtained at 293 K by the hanging-drop vapour-diffusion technique using the Nucleic Acid Mini Screen. Over a period of two weeks, hexagonal plates appeared. For the DNA fragment d(CCGGATCCGG), the crystals belong to space group P3(1), with unit-cell parameters a = b = 33.54, c = 46.39 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 2.2 A. In the case of the DNA fragment d(CCGGCGCCGG) the crystals belong to the space group C2, with unit-cell parameters a = 65.35, b = 24.07, c = 37.34 A, beta = 109.97 degrees, and diffract to 2.0 A.

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.

The layered organization of nucleosomes in 30 nm chromatin fibers.

We have used electron microscopy and established methods of three-dimensional reconstruction to obtain structural information on the 30 nm chromatin fibers from sea cucumber sperm and chicken erythrocytes. The fibers show a longitudinal periodicity of 10-11 nm. We have interpreted this periodicity as due to a grouping of nucleosomes into disks, each disk containing about 5-6 nucleosomes. These disks are closely stacked to form the chromatin fiber. We have built a detailed model for four fibers and we have determined the approximate coordinates of all the nucleosomes in them. The average distance found between neighboring nucleosomes has a value close to 11 nm. They may be connected either as a regularly distorted helix or as a layered zigzag. The second model appears more appropriate, since in the constrictions of the fibers the nucleosomes can only be connected as a zigzag.

Crystallization and preliminary X-ray analysis of the antimalarial and cytotoxic alkaloid cryptolepine complexed with the DNA fragment d(CCTAGG)2.

Crystals of the indoloquinoline alkaloid cryptolepine complexed with the DNA fragment d(CCTAGG)(2) have been grown by the hanging-drop technique at 293 K using ammonium sulfate as the precipitating agent. Over a period of three weeks, yellow tapering bullet-shaped crystals grew to maximum dimensions of 0.2 x 0.1 x 0.1 mm. The crystals belong to space group P6(4), with unit-cell parameters a = b = 29.960, c = 39.64 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 1.4 A.

The propeller DNA conformation of poly(dA).poly(dT).

Physical properties of the DNA duplex, poly(dA).poly(dT) differ considerably from the alternating copolymer poly(dAT). A number of molecular models have been used to describe these structures obtained from fiber X-ray diffraction data. The recent solutions of single crystal DNA dodecamer structures with segments of oligo-A.oligo-T have revealed the presence of a high propeller twist in the AT regions which is stabilized by the formation of bifurcated (three-center) hydrogen bonds on the floor of the major groove, involving the N6 amino group of adenine hydrogen bonding to two O4 atoms of adjacent thymine residues on the opposite strand. Here we show that it is possible to incorporate the features of the single crystal analysis, specifically high propeller twist, bifurcated hydrogen bonds, and a narrow minor groove, as well as the close interstrand NMR signal between adenine HC2 and ribose HC1' of the opposite strand, into a model that is fully compatible with the diffraction data obtained from poly(dA).poly(dT).

Crystal structure of a DNA Holliday junction.

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 A resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.

Effects of 5-fluorouracil/guanine wobble base pairs in Z-DNA: molecular and crystal structure of d(CGCGFG).

The chemotherapeutic agent 5-fluorouracil is a DNA base analogue which is known to incorporate into DNA in vivo. We have solved the structure of the oligonucleotide d(CGCGFG), where F is 5-fluorouracil (5FU). The DNA hexamer crystallizes in the Z-DNA conformation at two pH values with the 5FU forming a wobble base pair with guanine in both crystal forms. No evidence of the enol or ionized form of 5FU is found under either condition. The crystals diffracted X-rays to a resolution of 1.5 A and their structures have been refined to R-factors of 20.0% and 17.2%, respectively, for the pH = 7.0 and pH = 9.0 forms. By comparing this structure to that of d(CGCGCG) and d(CGCGTG), we were able to demonstrate that the backbone conformation of d(CGCGFG) is similar to that of the archetypal Z-DNA. The two F-G wobble base pairs in the duplex are structurally similar to the T-G base pairs both with respect to the DNA helix itself and its interactions with solvent molecules. In both cases water molecules associated with the wobble base pairs bridge between the bases and stabilize the structure. The fluorine in the 5FU base is hydrophobic and is not hydrogen bonded to any solvent molecules.

Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment.

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.

Crystal structure of a helical oligopeptide model of polyglycine II and of other polyamides: acetyl-(glycyl-beta-alanyl)2-NH propyl.

We synthesized and solved the crystalline structure of the oligopeptide acetyl-(glycyl-beta-alanyl)2-NH propyl. The crystal is formed by layers of helical molecules with the same chirality; however, right-handed layers alternate with left-handed ones. Inside every layer, the packing of helices is pseudohexagonal with hydrogen bonds between neighbor molecules. The structure found affords direct support for the model proposed by Crick and Rich for polyglycine II and also provides an interpretation for the structure of a newly found family of polyamides that do not form sheets as observed in most nylon structures.

Molecular structure of nicked DNA: a substrate for DNA repair enzymes.

The molecular structure of a nicked dodecamer DNA double helix, made of a ternary system containing d(CGCGAAAACGCG) + d(CGCGTT) + d(TTCGCG) oligonucleotides, has been determined by x-ray diffraction analysis at 3 A resolution. The molecule adopts a B-DNA conformation, not unlike those found in intact dodecamer DNA molecules crystallized in a somewhat different crystal lattice, despite a gap due to the absence of a phosphate group in the molecule. The helix has a distinct narrow minor groove near the center of the molecule at the AAAA region. This suggests that the internal stabilizing forces due to base stacking and hydrogen-bonding interactions are sufficient to overcome the loss of connectivity associated with the disruption of the covalent backbone of DNA.

Satellite DNAs contain sequences that induced curvature.

The repeating units of mouse, rat, and alpha-monkey satellites have been cloned. All three show properties that are characteristic of curved DNA: (i) their migration in polyacrylamide gels is slower than predicted from their sequences, and (ii) they appear as curved molecules when visualized by electron microscopy. All three satellite repeats contain runs of d(A.T)n greater than or equal to 3 residues that are likely to be responsible for their curvature. From analysis of 20 different satellite DNA sequences, we conclude that, in satellite DNA, adenine residues show a high tendency to cluster in groups of three or more.

Binding of a Hoechst dye to d(CGCGATATCGCG) and its influence on the conformation of the DNA fragment.

Hoechst dye 33258 is a planar drug molecule that binds to the minor groove of DNA, especially where there are a number of A.T base pairs. We have solved the structure of the Hoechst dye bound to the DNA dodecamer d(CGCGATATCGCG) at 2.3 A. This structure is compared to that of the same dodecamer with the minor-groove-binding drug netropsin bound to it, as well as to structures that have been solved for this Hoechst dye bound to a DNA dodecamer containing the central four base pairs with the sequence AATT. We find that the position of the Hoechst drug in this dodecamer is quite different from that found in the other dodecamer since it has an opposite orientation compared to the other two structures. The drug covers three of the four A.T base pairs and extends its piperazine ring to the first G.C base pair adjacent to the alternating AT segment. Furthermore, the drug binding has modified the structure of the DNA dodecamer. Other DNA dodecamers with alternating AT sequences show an alternation in the size of the helical twist between the ApT step (small twist) and the TpA step (large twist). In this structure the alternation is reversed with larger twists in the ApT steps than in the TpA step. In addition, there is a rotation of one of the thymine bases in the DNA dodecamer that is associated with hydrogen bonding to the Hoechst drug. This structure illustrates the considerable plasticity found in the DNA molecule when it binds to different planar molecules inserted into the minor groove.

Molecular structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor groove binding drug netropsin.

The molecular structure of the complex between the minor groove binding drug netropsin and the dodecamer d(CGCAAATTTGCG) has been solved and refined by X-ray diffraction analysis to an R-factor of 19.8% and 2.2-A resolution. The drug lies in the narrow minor groove of the B-DNA fragment, covering five of the six A.T base pairs (from A5.T20 to T9.A16). The long six A.T base pair tract allows the drug to bind in a position that optimizes its contacts with the DNA, establishing hydrogen bonds with O2 of thymines and N3 of adenines. The DNA molecule shows a high propeller twist only at the A6.T19 step of the A-tract. Two three-centered hydrogen bonds are observed in the major groove at half of the A-tract.

Three-dimensional crystal structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor-groove-binding drug Hoechst 33258.

The molecular structure of the DNA A-tract dodecamer d(CGCAAATTTGCG) complexed with the drug Hoechst 33258 has been determined by X-ray diffraction analysis. The Hoechst molecule binds in the DNA minor groove covering the sequence AATTT of the central A-tract, with the piperazine group close to one of the GC regions. The drug molecule makes two three-centered hydrogen bonds from the nitrogen atoms of the benzimidazole rings to the N3 and O2 atoms of the DNA bases. Although a high propeller twist is observed in the A-tract, only one unsymmetrical three-centered hydrogen bond is present in the DNA major groove. The structure is compared with other minor-groove-binding drug complexes and the influence of these drugs on DNA A-tracts is discussed.