Investigation of DNA Binding Modes for a Symmetrical Cyanine Dye Trication: Effect of DNA Sequence and Structure

Abstract

The DNA binding behavior of a tricationic cyanine dye (DiSC₃₊(5)) was studied using the [Poly(dA-dT)]₂, [Poly(dI-dC)]₂ and Poly(dA)?Poly(dT) duplex sequences and the Poly(dA) ?2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA)?Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]₂ duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]₂ sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987–2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

Uncertainty Evaluation of a Groundwater Conceptual Model by Using a Multimodel Averaging Method

ABSTRACT

A groundwater field is a complex and open system. Groundwater simulation and prediction often deviated from true values, which is attributed to the uncertainty of groundwater modeling. The conceptual model (model struture) is one of the main sources of groundwater modeling uncertianty. In this study, the mean Euclidean distance (MED) between model simulations and observations is proposed to assess the integrated likelihood value of a conceptual model in Bayesian model averaging (BMA). Moreover, this proposed BMA method is compared with the traditional generalized likelihood uncertainty estimation (GLUE) BMA method by a synthetical groundwater model, and the characteristics of these two BMA methods are summarized.     

X-Ray Crystallographic Studies of the A-Form of DNA

Abstract

The three-dimensional structure in the crystalline state of the self-complementary oligodeoxyribonucleotides d(GCCCGGGC) and d(GGGATCCC) is described and compared with other DNA fragments in the A-form.     

Sequence-Dependent Bending of DNA and Phasing of Nucleosomes

Abstract

Conformational analysis has revealed anisotropic flexibility of the B-DNA double helix: it bends most easily into the grooves, being the most rigid when bent in a perpendicular direction. This result implies that DNA in a nucleosome is curved by means of relatively sharp bends (“mini-kinks”) which are directed into the major and minor grooves alternatively and separated by 5–6 base pairs. The “mini-kink” model proved to be in keeping with the x-ray structure of the B-DNA dodecamer resolved later, which exhibits two “annealed kinks”, also directed into the grooves.

The anisotropy of B DNA is sequence-dependent: the pyrimidine-purine dimers (YR) favor bending into the minor groove, and the purine-pyrimidine dinucleotides (RY), into the minor one. The RR and YY dimers appear to be the most rigid dinucleotides. Thus, a DNA fragment consisting of the interchanging oligopurine and oligopyrmidine blocks 5–6 base pairs long should manifest a spectacular curvature in solution.

Similarly, a nucleotide sequence containing the RY and YR dimers separated by a half-pitch of the double helix is the most suitable for wrapping around the nucleosomal core. Analysis of the numerous examples demonstrating the specific alignment of nucleosomes on DNA confirms this concept. So, the sequence-dependent “mechanical” properties of the double helix influence the spatial arrangement of DNA in chromatin.     

NMR Studies of a DNA Containing 8-Methoxydeoxyguanosine

Abstract

¹H NMR experiments have been undertaken to elucidate the structural effects of methoxy substitution at the C8 of a deoxyguanosine residue in a self-complementary dodecadeoxyribonucleotide, d(C-G-C-mo⁸G-A-A-T-T-C-G-C-G), duplex, which has an 8-methoxy-2′-deoxyguanosine (mo⁸dG) residue at the 4th position. NMR data indicate that the mo⁸dG residue takes an anti glycosidic conformation in a mo⁸dG(anti):dC(anti) base-pair structure in a B-form duplex. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(C-G-C-G-A-A-T-T-C-G-C-G) duplex.     

Design of Sequence-Specific DNA Binding Ligands That Use a Two-Stranded Peptide Motif for DNA Sequence Recognition

Abstract

The design and DNA binding activity of β-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides - S,S'-bis(Lys-Gly-Val-Cys-Val- NH-NH-Dns) - bridged by an S-S bond binds at least 10 times more strongly to poly(dG)?poly(dC) than to poly(dA)?poly(dT). This peptide can also discriminate between 5′-GpG-3′ and 5′-GpC-3′ steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides - Gly-Cys-Gly- or Val-Cys-Val - bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5′-GpG-3′ steps on DNA, whereas the netropsin-like fragments bind preferentially to tuns of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5′-GpG-3′-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.     

A Kinked Model for the Solution Structure of DNA Tridecamers with Inserted Adenosines: Energy Minimization and Molecular Dynamics

Abstract

Structural modelling techniques using energy minimization and molecular dynamics have been employed to generate kinked models for the solution structure of two DNA tridecamer sequences containing inserted adenosines: d(CGCAGAATTCGCG)₂ and d(CGCAGAGCTCGCG)₂. These models are consistent with NMR studies of these sequences in solution. The overall shapes of the two models are similar, consisting of three B-DNA sections: two outer segments on the same side of the central portion, with the additional adenosines acting as wedges to kink the structure. An alternative scheme for the hydrogen bond pairing at the kink site is suggested as a way for the additional adenosines to be stabilized in the duplex.     

Crystal Structure of A Z-DNA Fragment Containing Thymine/2-Aminoadenine Base Pairs

Abstract

The Z-DNA structure has been shown to form in two crystals made from self-complementary DNA hexamers d(CGTDCG) and d(CDCGTG) which contain thymine/2-ammoadenine (TD) base pairs. The latter structure has been solved and refined to 1.3 Å resolution and it shows only small conformational changes due to the introduction of the TD base pairs in comparison with the structure of d(CG)3. Spectroscopic studies with these compounds demonstrate that DNA molecules containing 2-aminoadenine residues form Z-DNA slightly more easily than do those containing adenine nucleotides, but not as readily as the parent sequence containing only guanine-cytosine base pairs.     

Conformational and Model-Building Studies of the Hairpin Form of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G)

Abstract

The hairpin form of the mismatched octamer d(m⁵C-G-m⁵C-G-T-G-m⁵C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3′-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5′-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5′-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual γ^t and β⁺ torsion angles in residue dG(6).

The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone.

The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T- A-T₄-T-A-G-G-A-T), in which the central -T₄- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.     

Flexibility Of DNA In 2:1 Drug-DNA Complexes - Simultaneous Binding Of Two DAPI Molecules To DNA

Abstract

Simultaneous binding of two DAPI molecules in the minor groove of (dA)₁₅.(dT)₁₅ B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 Å) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.     

FTIR Study of Specific Binding Interactions Between DNA Minor Groove Binding Ligands and Polynucleotides

Abstract

The use of FTIR spectroscopy is made to study the interactions between polynucleotides and two series of minor groove binding compounds. The latter were developed and described previously as part of an ongoing program of rational design of modified Ligands based on naturally occurring pyrrole amidine antibiotic netropsin, and varying the structure of bis- benzimidazole chromosomal stain Hoechst 33258. Characteristic IR absorptions due to the vibrations of thymidine and cytosine keto groups in polynucleotides containing AT and GC base pairs respectively are used to monitor their interaction with the added Ligands. Although the two thiazole based lexitropsins based on netropsin structure differ in the relative orientation of nitrogen and sulfur atoms with respect to the concave edge of the molecules, they interact exclusively with the thymidine C2=O carbonyl groups in the minor groove of the alternating AT polymer as evidenced by specific changes in the IR spectra.

In the second series of compounds based on Hoechst 33258, the structure obtained by replacing the two benzimidazoles in the parent compound by a combination of pyridoimidazole and benzoxazole, exhibits changes in the carbonyl frequency region of poly dG · poly dC which is attributed to the ligand interaction at the minor groove of GC base pairs. In contrast, Hoechst 33258 itself interacts only with poly dA · poly dT. Weak or no interaction exists between the Ligands and any of the polynucleotides at the levels of the phosphate groups or the deoxyribose units.     

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

Abstract

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations.

As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be ?3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

1H-NMR Study of the Quadruplex [d(TGGGT)]4 Containing a Modified Thymine

Abstract

A NMR structural study of quadruplex [d(TGGGT)]₄ containing a modified thymine is reported. The three dimensional structure of the complex is very similar to those of other parallel stranded quadruplexes. The modified thymines (T*) are able, at least in the minimised structures, to form a tetrad containing extra H-bonds through the hydroxyl groups. Nevertheless, in this new tetrad the modified thymines are slightly open towards the solvent respect to the unmodified T-tetrad.     

Site-Specific 13C Labeling of DNA to Deduce DNA Repair Mechanisms of Uracil-DNA Glycosyiase and UV Endonuclease V

Abstract

The oligodeoxynucleotide d(GCGUGCG) was synthesized with [1′,3′ -¹³C₂)U labeling. The uracil unit was removed with uracil-DNA glycosylase to generate an abasic site and the resulting oligonucleotide was paired with the possible d(CGCNCGC) structures. One of these heteroduplexes was a substrate for W endonuclease V. The ¹³C NMR spectra of these heteroduplexes describe the structure of the abasic site and the mechanism of the endonuclease reaction.     

The IN-VIVO Occurrence of Z DNA

Abstract

The energetics of the B-Z transition of two different types of cloned alternating purine/pyrimidine DNA sequences have been analysed by a two dimensional electrophoretic technique. Since the transition between right handed and left handed forms of these polymers is detected by alterations of electrophoretic mobilities of topoisomers of the plasmid DNA molecules, the method is not dependent on Z-DNA binding ligands. The measurements reflect intrinsic properties of the DNA unperturbed by the free energy of binding such a ligand.

Direct evidence from the analysis of topoisomer distributions is presented which shows that d(GC)n.d(GC)n sequence elements within an E. coli plasmid will adopt a Z conformation in-vivo under conditions of blocked protein synthesis. Evidence for the in-vivo occurrence of Z-DNA was not detected in plasmid DNA isolated from bacterial cells growing in the absence of protein synthesis inhibitors.

A model is proposed for a function for the B-Z transition in ensuring the correct pairing of homologous chromosomes during meiosis.     

Structural Features and Hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a Double Helix Containing Two G.A Mispairs

Abstract

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5Å resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-l and 0–6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Conformations of DNA Duplexes Containing 8-Oxoguanine

Abstract

As a step towards elucidating the mechanisms of mutagenesis induced by irradiation and oxidation, we study the incorporation of 8-oxoguanine (OG) into duplex DNA. Molecular modelling is used to reveal changes in DNA conformational parameters due to mispairs within the sequences d(A₅XA₅) · d(T₅YT₅) and d(G₅XG₅) · d(C₅YC₅) where one of the bases of the bases of the central X:Y pair is OG and the other A T, G or C. The G:C to OG:C replacements in DNA duplexes produce only minor conformational changes, similar to normal base sequence effects. The calculations suggest that both OG(syn): G and OG(syn): A mispairs can also be introduced without drastic distortion of sugar-phosphate backbone. The distortions produced by OG-containing mispairs are also found to be sequence dependent Overall these calculations suggest that the G→OG conversion could be an important factor in the irradiative or oxidative damage of DNA.