Zippora Shakked

A hypothesis on a specific sequence-dependent conformation of DNA and its relation to the binding of the lac-repressor protein

The structure and properties of the double-helical form of the alternating copolymer poly(dA-dT) are considered. Different lines of evidence are interpreted in terms of a structure in which every second phosphate-diester linkage has a conformation different from that of the normal B form. A rationale for this “alternating-B” structure is given which provides an explanation for the effects of chemical modifications of the T residues on the binding of the poly(dA-dT)· poly(dA-dT) to the lac repressor of Escherichia coli.

Molecular flexibility in ab initio drug docking to DNA: binding-site and binding-mode transitions in all-atom Monte Carlo simulations

The dynamics of biological processes depend on the structure and flexibility of the interacting molecules. In particular, the conformational diversity of DNA allows for large deformations upon binding. Drug–DNA interactions are of high pharmaceutical interest since the mode of action of anticancer, antiviral, antibacterial and other drugs is directly associated with their binding to DNA. A reliable prediction of drug–DNA binding at the atomic level by molecular docking methods provides the basis for the design of new drug compounds. Here, we propose a novel Monte Carlo (MC) algorithm for drug–DNA docking that accounts for the molecular flexibility of both constituents and samples the docking geometry without any prior binding-site selection. The binding of the antimalarial drug methylene blue at the DNA minor groove with a preference of binding to AT-rich over GC-rich base sequences is obtained in MC simulations in accordance with experimental data. In addition, the transition between two drug–DNA-binding modes, intercalation and minor-groove binding, has been achieved in dependence on the DNA base sequence. The reliable ab initio prediction of drug–DNA binding achieved by our new MC docking algorithm is an important step towards a realistic description of the structure and dynamics of molecular recognition in biological systems.

Diversity in DNA recognition by p53 revealed by crystal structures with Hoogsteen base pairs

p53 binds as a tetramer to DNA targets consisting of two decameric half-sites separated by a variable spacer. Here we present high-resolution crystal structures of complexes between p53 core-domain tetramers and DNA targets consisting of contiguous half-sites. In contrast to previously reported p53–DNA complexes that show standard Watson-Crick base pairs, the newly reported structures show noncanonical Hoogsteen base-pairing geometry at the central A-T doublet of each half-site. Structural and computational analyses show that the Hoogsteen geometry distinctly modulates the B-DNA helix in terms of local shape and electrostatic potential, which, together with the contiguous DNA configuration, results in enhanced protein-DNA and protein-protein interactions compared to noncontiguous half-sites. Our results suggest a mechanism relating spacer length to protein-DNA binding affinity. Our findings also expand the current understanding of protein-DNA recognition and establish the structural and chemical properties of Hoogsteen base pairs as the basis for a novel mode of sequence readout.

DNA bending by an adenine–thymine tract and its role in gene regulation

To gain insight into the structural basis of DNA bending by adenine–thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2–DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein–DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.

The effect of the base sequence on the fine structure of the DNA double helix

Structural polymorphism of DNA has been studied for nearly thirty years by means of fibre diffraction techniques (Watson and Crick, 1953; Langridge et al., 1960; Arnott, 1976; Leslie et al., 1980). However, because of their limited resolution X-ray fibre diffraction patterns provide only an averaged conformation of the DNA and hence detection of local variations in the double helix induced by the particular base sequence is beyond the capability of this technique. A knowledge of the detailed structure of DNA became of increasing interest in recent years as evidence accumulated on sequence-specific recognition of DNA by various molecules such as enzymes and regulatory proteins on the one hand and antitumour drugs on the other. Structural studies on detailed sequence effects in DNA became feasible in the late 1970s when methods for DNA synthesis developed to the point where oligonucleotides could be produced in the quantity and purity needed for the preparation of single crystals for X-ray analysis. Unlike fibres, single crystals are ordered in three dimensions, can diffract to higher resolution and yield more data. These factors enable one to solve structures and observe fine details of DNA conformation. X-ray analyses of single crystals of deoxyoligonucleotides carried out in the past few years yielded detailed structural information on three forms: two right-handed structures similar in their overall conformations to the well established Aand B-DNA double helices derived by fibre methods (see reviews by Shakked and Kennard, 1985; Dickerson et al., 1985), and a radically new left-handed double-helical structure named Z-DNA and observed mainly in alternating sequences of purine and pyrimidine bases (see review by Wang and Rich, 1985). This left-handed form was also identified later in fibres of poly(dG-dC) (Arnott et al., 1980). In fibres, the transition between the various forms depends on external conditions such as relative humidity and salt content, as well as on the base sequence (Leslie et al., 1980). Crystallization, on the other hand, always involves dehydration processes, and although in several cases similar crystallization conditions were applied, three distinct forms were obtained. Crystal forces appear to have only small effects on the fine conformation of the double-helical fragments. Hence, it was felt that structural information obtained from highresolution X-ray studies should help in understanding the influence of the base sequence, rather than the external environment, on the structure of the DNA double helix. X-ray studies of complexes of DNA fragments with other molecules have been reported

A novel form of the DNA double helix imposed on the TATA-box by the TATA-binding protein

The structure of the TATA-box bound to the TATA-binding protein revealed a new and unexpected deformation of the double helix leading to a sharp change in the DNA trajectory. Here we show that the deformation imposed upon the TATA-box represents a novel form of the double helix—named TA-DNA—which differs from A-DNA by a single conformational parameter, namely the rotation around the glycosidic bond. This rotation causes a 50° inclination of the base pairs in the TATA-box which in turn results in abrupt change in the trajectory of the flanking B-DNA segments. The observation that the TATA sequence can assume an A-DNA conformation coupled to the simplicity of the transition from A-DNA to TA-DNA may be the reason for the presence of the TATA sequence in a wide range of promoters.

Ordered water structure in an A-DNA octamer at 1.7 A resolution.

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.

Crystalline A-DNA: the X-ray analysis of the fragment d(G-G-T-A-T-A-C-C)

An A-DNA type double helical conformation was observed in the single crystal X-ray structure of the octamer d(G-G-T-A-T-A-C-C), 1, and its 5-bromouracil-containing analogue, 2. The structure of the isomorphous crystals (space group P 61 was solved by a search technique based on packing criteria and R-factor calculations, with use of only low order data. At the present stage of refinement the R factors are 31% for 1 and 28% for 2 at a resolution of 2.25 Å (0.225 nm). The molecules interact through their minor grooves by hydrogen bonding and base to sugar van der Waals contacts. The stable A conformation observed in the crystal may have some structural relevance to promoter regions where the T-A-T-A sequence is frequently found.

The conformation of the DNA double helix in the crystal is dependent on its environment.

STUDIES of the crystal structures of more than 30 synthetic DNA fragments have provided structural information about three basic forms of the double helix: A-, B- and Z-form DNA1–5. These studies have demonstrated that the DNA double helix adopts a highly variable structure which is related to its base sequence. The extent to which such observed structures are influenced by the crystalline environment can be found by studying the same molecule in different crystalline forms. We have recently crystallized one particular oligomer in various crystal forms. Here we report the results of structural analyses of the different crystal structures and demonstrate that the DNA double helix can adopt a range of conformations in the crystalline state depending on hydration, molecular packing and temperature. These results have implications on our understanding of the influence of the environment on DNA structure, and on the modes of DNA recognition by proteins.

Structures of the mismatched duplex d(GGGTGCCC) and one of its Watson-Crick analogues d(GGGCGCCC).

The mismatched duplex d(GGGTGCCC) (I) and its two Watson-Crick analogues (dGGGCGCCC) (II) and d(GGGTACCC) (III) were synthesized. The X-ray crystal structures of (I) and (II) were determined at resolutions of 2.5 and 1.7 A (1 A = 0.1 nm) and refined to R factors of 15 and 16%, respectively. (I) and (II) crystallize as A-DNA doublehelical octamers in space groups P61 and P4(3)2(1)2, respectively, and are stable at room temperature. The central two G.T mispairs of (I) adopt the wobble geometry as observed in other G.T mismatches. The two structures differ significantly in their local conformational features at the central helical regions as well as in some global ones. In particular, T-G adopts a large helical twist (44 degrees) whereas C-G adopts a small one (24 degrees). This difference can be rationalized on the basis of simple geometrical considerations. Base-pair stacking energies which were calculated for the two duplexes indicate that (I) is destabilized with respect to (II). Helix-coil transition measurements were performed for each of the three oligomers by means of ultraviolet absorbance spectrophotometry. The results indicate that the stability of the duplexes and the co-operativity of the transition are in the following order: (I) less than (III) less than (II). Such studies may help in understanding why certain regions of DNA are more likely to undergo spontaneous mutations than others.