Variations in duplex DNA conformation detected by the binding of monoclonal autoimmune antibodies.

Four monoclonal antibodies (Jel 229, 239, 241, 242) which bound to duplex DNA were prepared from two autoimmune female NZB/NZW mice. Their binding to various nucleic acids was investigated by a competitive solid phase radioimmune assay which allows the estimation of relative binding constants. None of the antibodies showed any consistent variation of binding constant with base composition and thus they must recognize features of the DNA backbone. Jel 241 binds across the major groove but the interaction with poly(pyrimidine) X poly(purine) DNAs was barely detectable. This antibody appears to recognize the "alternating-B" conformation which is promoted by methylation of pyrimidines in alternating sequences. The other three antibodies bind in the minor groove. In particular, for Jel 229 the preferred antigen was poly(dG) X poly(dC) with only weak binding to poly(dA) X poly(dT). This suggests a requirement for a wide minor groove. Thus autoimmune antibodies provide examples of "analogue" recognition and can be used to detect structural variations in the grooves of duplex DNA.     

Molecular dynamics of spermine-DNA interactions: sequence specificity and DNA bending for a simple ligand.

We used molecular dynamics to model interactions between the physiologically important polyamine spermine and two B-DNA oligomers, the homopolymer (dG)10-(dC)10 and the heteropolymer (dGdC)5-(dGdC)5. Water and counterions were included in the simulation. Starting coordinates for spermine-DNA complexes were structures obtained by molecular mechanics modeling of spermine with the two oligomers; in these models, spermine binding induced a bend in the heteropolymer but not in the homopolymer. During approximately 40 psec of molecular dynamics simulation, spermine moves away from the floor of the major groove and interacts nospecifically with d(G)10-d(C)10. In contrast, a spermine-induced bend in the helix of (dGdC)5-(dGdC)5 is maintained throughout the simulation and spermine remains closely associated with the major groove. These results provide further evidence that the binding of spermine to nucleic acids can be sequence specific and that bending of alternating purine-pyrimidine sequences may be a physiologically important result of spermine binding.     

Importance of minor groove functional groups for the stability of DNA duplexes

Eight oligonucleotide duplexes have been prepared with four pairs of selected complementary pairs of native/analogue heterocyclic bases incorporated at a selected test site. The base pairs vary in the nature of their functionality in the minor groove. Each pair has a minor groove purine amino group present or absent, and correspondingly has a minor grove pyrimidine carbonyl present or absent. Loss of duplex stability is most notable when the minor groove pyrimidine carbonyl is absent although in other respects normal Watson-Crick hydrogen bonding is maintained in these sequences. These differences in stability are discussed in terms of possible variations in minor groove hydration.     

Structural Analysis of HMGD-DNA Complexes Reveals Influence of Intercalation on Sequence Selectivity and DNA Bending

The ubiquitous, eukaryotic, high-mobility group box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor-groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity, and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and 50 bp of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1 box A-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending.     

Effects of DNA looping on pyrimidine dimer formation.

We have assessed the effects of DNA curvature on pyrimidine dimer (PD) formation by examining the pattern of PD formation in DNA held in a loop by lambda repressor. The loop region was composed of diverse DNA sequences such that potential PD sites occurred throughout the loop. PD formation in the loop occurred with peaks at approximately 10 base intervals, just 3' of where the bending of the DNA was inferred to be toward the major groove. This relationship between the peaks and the DNA curvature is essentially identical to that observed in the nucleosome. This indicates that DNA curvature is the major source of the periodicity of PD formation in the nucleosome, and supports an earlier model of the conformation of nucleosomal DNA based on PD formation. DNA loops containing diverse sequences should be of general value for assessing the effects of DNA curvature on DNA modification by other agents used to probe DNA-protein interactions and DNA conformation.     

Base pair opening within B-DNA: free energy pathways for GC and AT pairs from umbrella sampling simulations

The conformational pathways and the free energy variations for base opening into the major and minor grooves of a B-DNA duplex are studied using umbrella sampling molecular dynamics simulations. We compare both GC and AT base pair opening within a double-stranded d(GAGAGAGAGAGAG)· d(CTCTCTCTCTCTC) oligomer, and we are also able to study the impact of opening on the conformational and dynamic properties of DNA and on the surrounding solvent. The results indicate a two-stage opening process with an initial coupling of the movements of the bases within the perturbed base pair. Major and minor groove pathways are energetically comparable in the case of the pyrimidine bases, but the major groove pathway is favored for the larger purine bases. Base opening is coupled to changes in specific backbone dihedrals and certain helical distortions, including untwisting and bending, although all these effects are dependent on the particular base involved. Partial opening also leads to well defined water bridging sites, which may play a role in stabilizing the perturbed base pairs.     

Minor groove to major groove, an unusual DNA sequence-dependent change in bend directionality by a distamycin dimer

DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin binding affinity, cooperativity, and stoichiometry with three target DNA sequences that have different sizes of alternating AT sites, ATAT, ATATA, and ATATAT, have been determined by mass spectrometry and surface plasmon resonance to help explain the conformational changes. The results show that distamycin binds strongly to and bends five or six AT base pair minor groove sites as a dimer with positive cooperativity, while it binds to ATAT as a weak, slightly anticooperative dimer. The bending direction was evaluated with an in phase A-tract reference sequence. Unlike other similar monomer minor groove binding compounds, such as netropsin, the distamycin dimer changes the directionality of the overall curvature away from the minor groove to the major groove. This distinct structural effect may allow designed distamycin derivatives to have selective therapeutic effects.     

Hydrogen and hydration of DNA and RNA oligonucleotides

In this paper, hydrogen bonding interaction and hydration in crystal structures of both DNA and RNA oligonucleotides are discussed. Their roles in the formation and stabilization of oligonucleotides have been covered. Details of the Watson-Crick base pairs G.C and A.U in DNA and RNA are illustrated. The geometry of the wobble (mismatched) G.U base pairs and the cis and almost trans conformations of the mismatched U.U base pairs in RNA is described. The difference in hydration of the Watson-Crick base pairs G.C, A.U and the wobble G.U in different sequences of codon-anticodon interaction in double helical molecules are indicative of the effect of hydration. The hydration patterns of the phosphate, the 2'-hydroxyl groups, the water bridges linking the phosphate group, N7 (purine) and N4 of Cs or O4 of Us in the major groove, the water bridges between the 2'-hydroxyl group and N3 (purine) and O2 (pyrimidine) in the minor groove are discussed.     

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.     

Electrostatic free energy landscapes for DNA helix bending

Nucleic acids are highly charged polyanionic molecules; thus, the ionic conditions are crucial for nucleic acid structural changes such as bending. We use the tightly bound ion theory, which explicitly accounts for the correlation and ensemble effects for counterions, to calculate the electrostatic free energy landscapes for DNA helix bending. The electrostatic free energy landscapes show that DNA bending energy is strongly dependent on ion concentration, valency, and size. In a Na⁺ solution, DNA bending is electrostatically unfavorable because of the strong charge repulsion on backbone. With the increase of the Na⁺ concentration, the electrostatic bending repulsion is reduced and thus the bending becomes less unfavorable. In contrast, in an Mg²⁺ solution, ion correlation induces a possible attractive force between the different parts of the helical strands, resulting in bending. The electrostatically most favorable and unfavorable bending directions are toward the major and minor grooves, respectively. Decreasing the size of the divalent ions enhances the electrostatic bending attraction, causing an increased bending angle, and shifts the most favorable bending to the direction toward the minor groove. The microscopic analysis on ion-binding distribution reveals that the divalent ion-induced helix bending attraction may come from the correlated distribution of the ions across the grooves in the bending direction.     

A molecular dynamics study of the effect of G.T mispairs on the conformation of DNA in solution

The effect of G.T mispair incorporation into a double-helical environment was examined by molecular dynamics simulation. The 60-ps simulations performed on the two hexanucleotide duplexes d (G3C3)2 and d(G3TC2)2 included 10 Na+ counterions and first hydration shell waters. The resulting backbone torsional angle trajectories were analyzed to select time spans representative of conformational domains. The average backbone angles and helical parameters of the last time span for both duplexes are reported. During the simulation the hexamers retained B-type DNA structures that differed from typical A- or B-DNA forms. The overall helical structures for the two duplexes are vary similar. The presence of G.T mispairs did not alter the overall helical structure of the oligonucleotide duplex. Large propeller twist and buckle angles were obtained for both duplexes. The purine/pyrimidine crossover step showed a large decrease in propeller twist in the normal duplex but not in the mismatch duplex. Upon the formation of wobble mispairs in the mismatched duplex, the guanines moved into the minor groove and the thymines moved into the major groove. This helped prevent purine/purine clash and created a deformation in the relative orientation of the glycosidic bonds. It also exposed the free O4 of the thymines in the major groove and N2 of the guanines in the minor groove to interactions with solvent and counterions. These factors seemed to contribute to the apparently higher rigidity of the mismatched duplex during the simulation.     

1 A crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium

The 1 A resolution X-ray crystal structures of Mg(2+) and Ca(2+) salts of the B-DNA decamers CCAACGTTGG and CCAGCGCTGG reveal sequence-specific binding of Mg(2+) and Ca(2+) to the major and minor grooves of DNA, as well as non-specific binding to backbone phosphate oxygen atoms. Minor groove binding involves H-bond interactions between cross-strand DNA base atoms of adjacent base-pairs and the cations' water ligands. In the major groove the cations' water ligands can interact through H-bonds with O and N atoms from either one base or adjacent bases, and in addition the softer Ca(2+) can form polar covalent bonds bridging adjacent N7 and O6 atoms at GG bases. For reasons outlined earlier, localized monovalent cations are neither expected nor found.Ultra-high atomic resolution gives an unprecedented view of hydration in both grooves of DNA, permits an analysis of individual anisotropic displacement parameters, and reveals up to 22 divalent cations per DNA duplex. Each DNA helix is quite anisotropic, and alternate conformations, with motion in the direction of opening and closing the minor groove, are observed for the sugar-phosphate backbone. Taking into consideration the variability of experimental parameters and crystal packing environments among these four helices, and 24 other Mg(2+) and Ca(2+) bound B-DNA structures, we conclude that sequence-specific and strand-specific binding of Mg(2+) and Ca(2+) to the major groove causes DNA bending by base-roll compression towards the major groove, while sequence-specific binding of Mg(2+) and Ca(2+) in the minor groove has a negligible effect on helix curvature. The minor groove opens and closes to accommodate Mg(2+) and Ca(2+) without the necessity for significant bending of the overall helix.The program Shelxdna was written to facilitate refinement and analysis of X-ray crystal structures by Shelxl-97 and to plot and analyze one or more Curves and Freehelix output files.     

DNA Sequence Determinants Controlling Affinity,Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.     

Extra-helical guanine interactions in DNA

We review the extra-helical guanine interactions present in many oligonucleotide crystals. Very often terminal guanines interact with other guanines in the minor groove of neighboring oligonucleotides through N2 x N3 hydrogen bonds. In other cases the interaction occurs with the help of Ni2+ ions. Guanine/netropsin stacking in the minor groove has also been found. From these studies we conclude that guanine may have multiple extra-helical interactions. In particular it may be considered a very effective minor groove binder, which could be used in the design of sequence selective binding drugs. Interactions through the major groove are seldom encountered, but might be present when DNA is stretched. Such interactions are also analyzed, since they might be important for homologous chromosome pairing during meiosis.     

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.     

Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation.

The crystal structure of the HincII restriction endonuclease-DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine-purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base-stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.     

Sequences that adopt non-B-DNA conformation in form V DNA as probed by enzymic methylation

pBR322 form V DNA is a highly torsionally strained molecule with a linking number of zero. We have used sequence-specific DNA methylases as probes for B-DNA in this molecule, exploiting the inability of methylases to methylate single-stranded DNA and Z-DNA, both of which are known to occur in form V DNA. Some sequences in form V DNA were shown to be totally in the B-form, others were totally in an altered, unmethylatable conformation, while still other sites appeared to exist partly in altered and partly in normal B-conformation. Some potential Z-forming sequences (alternating pyrimidine/purine) of less than seven base-pairs were not in the Z conformation in form V DNA, whereas others did adopt an altered structure, indicating a modulating influence of flanking sequences. Furthermore, regions of imperfect alternating pyrimidine/purine structure were sometimes capable of adopting an altered structure. In addition, some regions of altered structure had no apparent Z-forming sequences, nor were they in polypurine stretches, which have also been proposed to form left-handed DNA. These non-B-DNA conformations may represent novel left-handed helical structures or sequences that become single stranded under torsional strain. Long regions of either altered (unmethylatable) DNA or B-DNA were not always observed. In fact, one region showed three transitions between B-like DNA and altered structure within 26 base-pairs.     

Ternary interactions of spermine with DNA: 4''-epiadriamycin and other DNA: anthracycline complexes.

The recently developed anthracycline 4'-epiadriamycin, an anti-cancer drug with improved activity, differs from adriamycin by inversion of the stereochemistry at the 4'-position. We have cocrystallized 4'-epiadriamycin with the DNA hexamer d(CGATCG) and solved the structure to 1.5 A resolution using x-ray crystallography. One drug molecule binds at each d(CG) step of the hexamer duplex. The anthracycline sugar binds in the minor groove. A feature of this complex which distinguishes it from the earlier DNA:adriamycin complex is a direct hydrogen bond from the 4'-hydroxyl group of the anthracycline sugar to the adenine N3 on the floor of the DNA minor groove. This hydrogen bond results directly from inversion of the stereochemistry at the 4'-position. Spermine molecules bind in the major groove of this complex. In anthracycline complexes with d(CGATCG) a spermine molecule binds to a continuous hydrophobic zone formed by the 5-methyl and C6 of a thymidine, C5 and C6 of a cytidine and the chromophore of the anthracycline. This report discusses three anthracycline complexes with d(CGATCG) in which the spermine molecules have different conformations yet form extensive van der Waals contacts with the same hydrophobic zone. Our results suggest that these hydrophobic interactions of spermine are DNA sequence specific and provide insight into the question of whether DNA:spermine complexes are delocalized and dynamic or site-specific and static.     

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.     

TIT for TAT: The Properties of Inosine and Adenosine in TATA Box DNA

Abstract

The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counteri-ons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.