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Biomedical subjects

R E Dickerson

Publications and source records attributed to R E Dickerson.

At least 73 records · Page 4Linked to original sources
PubMed1989

The structure of DAPI bound to DNA.

The structure of the DNA fluorochrome 4'-6-diamidine-2-phenyl indole (DAPI) bound to the synthetic B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G-C-G has been solved by single crystal x-ray diffraction methods, at a resolution of 2.4 A. The structure is nearly isomorphous with that of the native DNA molecule alone. With one DAPI and 25 waters per DNA double helix, the residual error is 21.5% for the 2428 reflections above the 2-sigma level. DAPI inserts itself edgewise into the narrow minor groove, displacing the ordered spine of hydration. DAPI and a single water molecule together span the four AT base pairs at the center of the duplex. The indole nitrogen forms a bifurcated hydrogen bond with the thymine O2 atoms of the two central base pairs, as with netropsin and Hoechst 33258. The preference of all three of these drugs for AT regions of B-DNA is a consequence of three factors: (1) The intrinsically narrower minor groove in AT regions than in GC regions of B-DNA, leading to a snug fit of the flat aromatic drug rings between the walls of the groove. (2) The more negative electrostatic potential within the minor groove in AT regions, attributable in part to the absence of electropositive-NH2 groups along the floor of the groove, and (3) The steric advantage of the absence of those same guanine-NH2 groups, thus permitting the drug molecule to sink deeper into the groove. Groove width and electrostatic factors are regional, and define the relative receptiveness of a section of DNA since they operate over several contiguous base pairs. The steric factor is local, varying from one base pair to the next, and hence is the means of fine-tuning sequence specificity.

Base Sequence↗
PubMed1988

X-ray structure of a DNA hairpin molecule.

We have solved the crystal structure of a synthetic DNA hexadecanucleotide of sequence: C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G, at 2.1 A resolution, and observed that it adopts a monomeric hairpin configuration with a Z-DNA hexamer stem. In the T4 loop the bases stack with one another and with neighbouring molecules of the crystal, and not with base pairs of their own hexamer stem. Two thymine T10 rings from different molecules stack between the C1-G16 ends of a third and a fourth hairpin helix, in a manner that suggests T-T base 'pairing' and simulates a long, 13-base-pair helix. Although such T-T interactions would not be present in solution, they illustrate a remarkable tendency of thymines for self-association. Purine-purine G-A base pairs are known to exist in the anti-anti conformation with an increase in local helix width; it may be that more serious consideration should be given to the possible existence of pyrimidine-pyrimidine C-T base pairs with decreased local helix width, particularly where several such base pairs occur sequentially.

Crystallography↗
PubMed1988

Structure of an alternating-B DNA helix and its relationship to A-tract DNA.

The crystal structure of the synthetic DNA dodecamer CGCATATATGCG has been solved at 2.2-A resolution. Its central 6 base pairs adopt the alternating-B-DNA helix structure proposed nearly a decade ago. This alternating poly(AT) structure contrasts with the four known examples of what can be termed a poly(A) subfamily of B-DNA structures: CGCAAAAAAGCG, CGCAAATTTGCG, CGCGAATTCGCG, and CGCGAATTbrCGCG, their defining characteristic being a succession of two or more adenines along one strand, in a region of 4 or more A.T base pairs. All five helices show a characteristically narrow minor groove in their AT centers, but the mean propeller twist at A.T base pairs is lower in the alternating poly(AT) helix than in the poly(A) subfamily of helices. Three general principles emerge from x-ray analyses of B-DNA oligonucleotides: (i) GC and mixed-sequence B-DNA have a wide minor groove, whereas the minor groove is narrow in heteropolymer or homopolymer AT sequences. (ii) G.C base pairs have low propeller twist; A.T pairs can adopt a high propeller twist but need not do so. A high propeller twist can be stabilized by cross-strand hydrogen bonds in the major or minor groove, examples being the minor groove bonds seen in CCAAGATTGG and the major groove bonds that can accompany AA sequences in the poly(A) family. (iii) Homopolymer poly(A) tracts may be stiffer than are alternating AT or general-sequence DNA because of these cross-strand major groove hydrogen bonds. Poly(A) tracts appear internally unbent, but bends may occur at junctions with mixed-sequence DNA because of differences in propeller twist, base pair inclination, and base stacking on the two sides of the junction. Bending occurs most easily via base roll, favoring compression of the broad major groove.

Base Composition↗
PubMed1987

Helix geometry, hydration, and G.A mismatch in a B-DNA decamer.

The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.

Base Composition↗
PubMed1987

Binding of Hoechst 33258 to the minor groove of B-DNA.

An X-ray crystallographic structure analysis has been carried out on the complex between the antibiotic and DNA fluorochrome Hoechst 33258 and a synthetic B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G. The drug molecule, which can be schematized as: phenol-benzimidazole-benzimidazole-piperazine, sits within the minor groove in the A-T-T-C region of the DNA double helix, displacing the spine of hydration that is found in drug-free DNA. The NH groups of the benzimidazoles make bridging three-center hydrogen bonds between adenine N-3 and thymine O-2 atoms on the edges of base-pairs, in a manner both mimicking the spine of hydration and calling to mind the binding of the auti-tumor drug netropsin. Two conformers of Hoechst are seen in roughly equal populations, related by 180 degrees rotation about the central benzimidazole-benzimidazole bond: one form in which the piperazine ring extends out from the surface of the double helix, and another in which it is buried deep within the minor groove. Steric clash between the drug and DNA dictates that the phenol-benzimidazole-benzimidazole portion of Hoechst 33258 binds only to A.T regions of DNA, whereas the piperazine ring demands the wider groove characteristic of G.C regions. Hence, the piperazine ring suggests a possible G.C-reading element for synthetic DNA sequence-reading drug analogs.

Base Sequence↗
PubMed1987

The effect of crystal packing on oligonucleotide double helix structure.

One of the questions that constantly is asked regarding x-ray crystal structure analyses of macromolecules is: To what extent is the observed crystal structure representative of the molecular conformation when free in solution, and to what degree is the structure perturbed by intermolecular crystal forces? This can be assessed with DNA oligomers because of an unusual aspect of crystallization self-complementary oligomers should possess a twofold symmetry axis normal to their helix axis, yet more often than not crystal of such oligomers do not use this internal symmetry. The two ends of the helix are crystallographically distinct though chemically identical. Complexes of DNA oligomers with intercalating drugs such as triostin A tend to use their twofold symmetry when they crystallize, whereas complexes with non-intercalating, groove-binding drugs ignore this symmetry unless the drug molecule is very small. A detailed examination of crystal packing in the dodecamer C-G-C-G-A-A-T-T-C-G-C-G provides an explanation of all of the foregoing behavior in terms of the mechanism of nucleation of DNA or DNA-drug complexes on the surface of a growing crystal. Asymmetry of the ends of the DNA helix is the price that is paid for efficient lateral packing of helices within the crystal. The actual end-for-end variation in standard helix parameters is compared with the experimental noise level as gauged by independent re-refinement of the same oligonucleotide structure where available, and with the observed extent of variation of these same parameters along the helix. Oligomers analyzed are the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, the A-DNA octamer G-G-T-A-T-A-C-C, and the phosphorothioate analogue of the B-DNA hexamer G-C-G-C-G-C. End-for-end variation, presumably the result of crystal packing is typically double the experimental noise level, and half the variation in the same parameter along the helix. Analysis of crystal packing in the phosphorothioate hexamer, which uses the same P212121 space group as the dodecamer, shows that the highly unsymmetrical B1 vs. BII backbone conformation probably is to be ascribed to crystal packing forces, and not to the sequence of the hexamer.

Antineoplastic Agents↗
PubMed1986

NMR study of a synthetic DNA hairpin.

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.

Base Composition↗
PubMed1986

Isohelical analysis of DNA groove-binding drugs.

Many antitumor drugs, and many carcinogens, act by binding within the minor groove of double-helical DNA, interfering with both replication and transcription. Several of these, including netropsin and distamycin, are quite base specific, recognizing and binding only to certain base sequences. The repeating pyrrole-amide unit of netropsin, and the repeated benzimidazole unit of the DNA stain and carcinogen Hoechst 33258, both are approximately 20% too long for synchronous meshing with base pairs along the floor of the minor groove in B DNA. We have carried out a systematic computer search for possible repeating drug backbones that are isohelical with DNA and that also provide chemical groups capable of reading and differentiating between A X T and G X C base pairs. These isohelical sequence-reading drug polymers or "isolexins" should offer the possibility of targeting synthetic drug analogues specifically against one region of a genome rather than another, or against neoplastic cells in preference to normal cells.

Bisbenzimidazole↗
PubMed1985

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G.

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.

Base Sequence↗
PubMed1985

The molecular origin of DNA-drug specificity in netropsin and distamycin.

X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-BrC-G-C-G reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH furnish hydrogen bonds to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 A, and it bends back the helix axis by 8 degrees across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to runs of four or more A X T base pairs is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G X C base pairs as well, and it could lead to a class of synthetic "lexitropsins," capable of reading any desired short sequence of DNA base pairs.

Base Sequence↗
PubMed1985

Nuclear Overhauser data and stereochemical considerations suggest that netropsin binds symmetrically within the minor groove of poly(dA).poly(dT), forming hydrogen bonds with both strands of the double helix.

Sarma et al. (J. Biomol. Str. and Dynam. 2, 1085 (1985) have proposed, on the basis of nuclear magnetic resonance experiments on the complex of netropsin with poly(dA).poly(dT), that the drug molecule lies asymmetrically along the dA side of the minor groove and makes hydrogen bonds only with the dA strand. If the crystal structure analyses of B-DNA (Fratini et al., J. Biol. Chem. 257, 14686 (1982] and of its complex with netropsin (Kopka et al., J. Mol. Biol. 183, 553 (1985] are any guide, this off-center, wide-groove model is stereochemically unlikely. More to the point, the off-center model is unnecessary to explain the observed nmr data. All of the nuclear Overhauser and other observations are fully explained by the structure seen in the x-ray crystal analysis, in which netropsin sits squarely centered within the minor groove, making bifurcated hydrogen bonds with both strands.

Binding Sites↗
PubMed1984

The kinetics of phosphotriester condensation reactions on solid support.

A study has been made of the kinetics of condensation reactions in phosphotriester synthesis of oligonucleotides on polystyrene copolymer resin support, using monomers, dimers, and trimers as the coupling units. The condensations follow pseudo first order kinetics, with a rate constant that decreases from monomers to dimers to trimers. Arrhenius plots indicate a nearly constant activation energy of around 10.4 kcal mol-1. Extrapolation to find the Arrhenius preexponential factor, A, is inaccurate because of the limited temperature range studied. But if the assumption is made that Ea is the same for monomer, dimer, and trimer reactions, then the A values are inversely proportional to the surface areas of the coupling units, reinforcing the interpretation of A as a collision efficiency factor.

Indicators and Reagents↗
PubMed1984

Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G.

The DNA oligomer of sequence IC-C-G-G has been synthesized, and its X-ray crystal structure solved at a resolution of 2.0 A, using anomalous scattering from iodines in phase analysis: 48 cycles of Jack-Levitt restrained least-squares refinement resulted in a residual error of 19.9% over all data, or 16.5% for two-sigma data. Two double-helical tetramers stack in the crystal to form a continuous octamer, except for the two missing phosphate connections across the center. The octamer has a mean helix rotation of 33.7 degrees (10.7 base-pairs per turn), rise of 2.87 A, mean inclination angle of base-pairs of 14 degrees, and mean base-pair propeller twist of +16.3 degrees. Local variations in both helix rotation and base plane roll angles, including those across the center of the octamer, are as predicted from base sequence by sum functions sigma 1 and sigma 2. The three known DNA octamers: IC-C-G-G/IC-C-G-G, G-G-T-A-T-A-C-C and G-G-C-C-G-G-C-C, make up a graded series in this order, with monotonically changing structural parameters. An exhaustive comparison of torsion angle correlations among the known A helices confirms some structural expectations and reveals some new features. 86 water molecules have been located per double-helical IC-C-G-G tetramer (the asymmetric unit), of which 451/2 per tetramer lie within a first hydrogen-bonded shell of hydration. No ordered water structure is observed comparable to the minor groove spine of hydration in B-DNA.

Base Sequence↗
PubMed1984

The primary mode of binding of cisplatin to a B-DNA dodecamer: C-G-C-G-A-A-T-T-C-G-C-G.

When cisplatin [cis- diamminodichloroplatinum (II)] is diffused into pre-grown crystals of the B-DNA double-helical dodecamer C-G-C-G-A-A-T-T-C-G-C-G, it binds preferentially to the N7 positions of guanines, with what probably is an aquo bridge between Pt and the adjacent O6 atom of the same guanine. The entire guanine ring moves slightly toward the platinum site, into the major groove. Only three of the eight potential cisplatin binding sites on guanines actually are occupied, and this differential reactivity can be explained in terms of the relative freedom of motion of guanines toward the major groove. This shift of guanines upon ligation may weaken the glycosyl bond and assist in the depurination that leads to mismatch SOS repair and G.C. to T.A. transversion.

Base Sequence↗
PubMed1983

Base sequence and helix structure variation in B and A DNA.

The observed propeller twist in base-pairs of crystalline double-helical DNA oligomers improves the stacking overlap along each individual helix strand. But, as proposed by Calladine, it also leads to clash or steric hindrance between purines at adjacent base-pairs on opposite strands of the helix. This clash can be relieved by: (1) decreasing the local helix twist angle between base-pairs; (2) opening up the roll angle between base-pairs on the side on which the clash occurs; (3) separating purines by sliding base-pairs along their long axes so that the purines are partially pulled out of the stack (leading to equal but opposite alterations in main-chain torsion angle delta at the two ends of the base-pair); and (4) flattening the propeller twist of the offending base-pairs. Simple sum functions, sigma 1 through sigma 4, are defined, by which the expected local variation in helix twist, base roll angle, torsion angle delta and propeller twist may be calculated from base sequence. All four functions are quite successful in predicting the behavior of B DNA. Only the helix twist and base roll functions are applicable to A DNA, and the helix twist function begins to fail for an A helical RNA/DNA hybrid. Within these limits, the sequence-derived sum functions match the observed helix parameter variation quite closely, with correlation coefficients greater than 0.900 in nearly all cases. Implications of this sequence-derived helix parameter variation for repressor-operator interactions are considered.

Base Composition↗