DNA interaction with the antitumor agent thiophosphamide

DNA interaction with an alkylating antitumor drug N,N',N"-triethylenethiophosphoramide (thiotepa) in water-salt solutions at 37 degrees C has been studied by UV-spectroscopy, heat denaturation and electron microscopy methods. Changes of the DNA melting curve parameters provide information on the kinetics of alkylation. The dependence of the alkylation rate on DNA and thiotepa concentrations shows that the alkylation reaction is biomolecular. The increase of sodium chloride concentration from 10(-3) to 10(-1) M is accompanied by a drastic decrease of the alkylation rate. Thiotepa binding results in destabilization of the DNA secondary structure and formation of cross-links. An increased amount of bounded thiotepa results in DNA denaturation; prolonged alkylation causes breaks in the sugar-phosphate backbone. The results of the work are discussed in connection with the literature data on DNA interaction with thiotepa in vivo.     

DNA minor groove electrostatic potential: influence of sequence-specific transitions of the torsion angle gamma and deoxyribose conformations

The structural adjustments of the sugar-phosphate DNA backbone (switching of the γ angle (O5′–C5′–C4′–C3′) from canonical to alternative conformations and/or C2′-endo → C3′-endo transition of deoxyribose) lead to the sequence-specific changes in accessible surface area of both polar and non-polar atoms of the grooves and the polar/hydrophobic profile of the latter ones. The distribution of the minor groove electrostatic potential is likely to be changing as a result of such conformational rearrangements in sugar-phosphate DNA backbone. Our analysis of the crystal structures of the short free DNA fragments and calculation of their electrostatic potentials allowed us to determine: (1) the number of classical and alternative γ angle conformations in the free B-DNA; (2) changes in the minor groove electrostatic potential, depending on the conformation of the sugar-phosphate DNA backbone; (3) the effect of the DNA sequence on the minor groove electrostatic potential. We have demonstrated that the structural adjustments of the DNA double helix (the conformations of the sugar-phosphate backbone and the minor groove dimensions) induce changes in the distribution of the minor groove electrostatic potential and are sequence-specific. Therefore, these features of the minor groove sizes and distribution of minor groove electrostatic potential can be used as a signal for recognition of the target DNA sequence by protein in the implementation of the indirect readout mechanism.     

The mechanics of minor groove width variation in DNA, and its implications for the accommodation of ligands.

In duplex DNA, groove width and depth are salient structural features that may influence the binding of drugs and proteins. These features are affected by movement of the bases, which for example may enforce groove compression or expansion through a rolling action of the adjacent base-pairs. Moreover, the sugar-phosphate backbone can also undergo limited movement, independently of the bases, which will affect the groove shape. We have examined how the movement of the sugar-phosphate backbone may affect the minor groove width for a fixed base geometry. In agreement with earlier studies, the sugar-phosphate backbone is found to have a certain degree of conformational flexibility in A and B-like helices, and we note a comparable freedom even in the highly curved TATA element of the TATA-binding protein/DNA complex. Phosphate mobility is highly anisotropic in all cases with favoured directions that can significantly change the groove width, independent of any changes in base geometry. We describe how the movement of the sugar-phosphate backbone may affect the accommodation of drugs and proteins in the minor groove, and we present a co-ordinate scheme which emphasises the groove adjustments associated with ligand binding. The observations have implications for the related problem of how cognate molecules are accommodated in the major groove.     

NMR studies of backbone-alkylated DNA: duplex stability, absolute stereochemistry, and chemical shift anomalies of prototypal isopropyl phosphotriester modified octanucleotides, (Rp,Rp)- and (Sp,Sp)-(d-[GGA(iPr)ATTCC])2 and -(d-[GGAA(iPr)TTCC])2

The DNA octamer (d-[GGAATTCC])2 and four alkylated analogues, (Rp)-(d-[GGA(iPr)ATTCC])2, (Sp)-(d-[GGA(iPr)ATTCC])2, (Rp)-(d-[GGAA(iPr)TTCC])2, and (Sp)-(d-[GGAA(iPr)TTCC])2 have been examined using 1H and 31PNMR spectroscopies. Duplex stability, as monitored by both NMR and optical measurements, is shown to be a function of both site and stereochemistry of the phosphotriester moiety. Chemical shift changes relative to the native octamer indicate that there are long-range perturbations in the isopropylated molecules. 1HNMR is shown to be a general means by which stereochemistry at phosphorous can be determined.     

The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide.

Both cyanogen bromide (BrCN) and 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide may be used as coupling reagents for the template-directed assembly of DNA duplexes containing the sugar-phosphate backbone modification. Both reagents show similar ligation site structure-specific trend. Practical recommendations are given for selection of the condensing reagent depending on the properties of the duplex. Based on 31P NMR spectroscopy data, a scheme is suggested for BrCN activation of the nucleotide phosphomonoester group. Using both condensing reagents, we studied the condensation of oligonucleotides containing ribo-segments (from mononucleotide residue to full sequence) on the DNA template. Efficiency of the chemical ligation of RNA oligomers was shown to be much lower than that of DNA analogues. The coupling yield depends on the position of the RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick.     

Energy bands and electronic delocalization in the sugar-phosphate backbone of DNA

The results of a CNDO/2 all-valence electron crystal orbital study are reported for the sugar-phosphate chain of DNA. The valence and conduction bandwidths are found to be large enough to make electronic delocalization through this backbone possible. Different mechanisms for charge carrier transport in DNA are compared on the basis of the electron and hole effective masses. Conduction along the backbone seems to be at least as probable as through the aperiodic system of the superimposed nucleotide bases.     

Investigation of the possibility of solitary waves in the base stacks of DNA.

The possibility of the existence of solitary waves in DNA is investigated. On the basis of our classical model we do not find such a wave in a polynucleotide, but for a stack of adenine molecules without backbone we observe one. Possible extensions of the model for DNA are discussed. From our results we can conclude, that solitons exist in stacked systems without an additional backbone. At least the degree of freedom which couples a nucleotide base (pair) to the sugar-phosphate backbones (N-C stretching vibration) has to be treated with the help of the quantum equations of motion.     

Flexibility of DNA in 2:1 drug-DNA complexes--simultaneous binding of two DAPI molecules to DNA.

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 angstroms) 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.     

Mutagenesis directed by phosphotriester analogs of oligonucleotides: a way to site-specific mutagenesis in vivo

A new approach is proposed to obtain the directed mutations in the gene under study. The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens. The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.     

NMR characterization of clustered bistrand abasic site lesions: effect of orientation on their solution structure

A unique characteristic of ionizing radiation and radiomimetic anticancer drugs is the induction of clustered damage: two or more DNA lesions (oxidized bases, abasic sites, or strand breaks) occurring in the same or different strands of the DNA molecule within a single turn of the helix. In spite of arising at a lower frequency than single lesions, clustered DNA damage represents an exotic challenge to the repair systems present in the cells and, in some cases, these lesions may escape detection and/or processing. To understand the structural properties of clustered DNA lesions we have prepared two oligodeoxynucleotide duplexes containing adjacent tetrahydrofuran residues (abasic site analogues), positioned one in each strand of the duplex in a 5' or 3' orientation, and determined their solution structure by NMR spectroscopy and molecular dynamics simulations. The NMR data indicate that both duplex structures are right-handed helices of high similarity outside the clustered damage site. The thermal stability of the duplexes is severely reduced by the presence of the abasic residues, especially in a 5' orientation where the melting temperature is 5 degrees C lower. The structures show remarkable differences at the lesion site where the extrahelical location of the tetrahydrofuran residues in the (AP)(2)-5'-staggered duplex contrasts with their smooth alignment along the sugar-phosphate backbone in the (AP)(2)-3'-staggered duplex.     

Reversible covalent modification of DNA

The reaction of bromomethylbenzoyl esters of choline and dimethylaminoethanol with DNA and model compounds led predominantly to phosphotriester formation. In model compounds the phosphotriester formation was verified by uv spectrometry. The bromomethylbenzoyl cationic esters reacted with DNA at room temperature at neutral pH values. The amount of the reagent chromophores was assessed semiquantitatively by spectrophotometry. The maximum binding appeared to be stoichiometric, i.e., one residue per phosphorus. The binding of one mole of reagent per phosphorus was confirmed by electron spectroscopic measurements of the phosphorus atom electron emission of maximally modified DNA. The modified DNA showed altered CD spectra indicating that the reagent chromophores are arranged in an orderly fashion affording a strong (Δ_? > 4), positive, apparently extrinsic CD band at ~240 nm; a double helical array is proposed. The introduced chromophores were readily removed by heat treatment or by treatment with nucleophiles at neutral pH values at moderate temperatures (<37 °C); no measurable fraction of the DNA became dialyzable. A decrease in viscosity accompanied the reversal, indicative of some chain breaking. The modified DNA's show higher T_m values than the native DNA and some display a higher and some a lower degree of cooperativity in their melting curves. No chemically detectable amounts of base alkylation, depurination, or depyrimidination were found when dialyzates of treated DNA and hydrolyzed samples of modified DNA were examined. However, presumptive evidence for some base alkylation by these novel alkylating agents was found utilizing Salmonella typhimurium tester strains sensitive to reversion by alkylation. No comparable binding of benzoylcholine, a nonalkylating analogue, by DNA was seen under conditions utilized here.     

Structure of DNA polymerase beta with the mutagenic DNA lesion 8-oxodeoxyguanine reveals structural insights into its coding potential

Oxidative damage to DNA generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). During DNA replication and repair synthesis, 8-oxodG can pair with cytosine or adenine. The ability to accurately replicate through this lesion depends on the DNA polymerase. We report the first structure of a polymerase with a promutagenic DNA lesion, 8-oxodG, in the confines of its active site. The modified guanine residue is in an anti conformation and forms Watson-Crick hydrogen bonds with an incoming dCTP. To accommodate the oxygen at C8, the 5'-phosphate backbone of the templating nucleotide flips 180 degrees. Thus, the flexibility of the template sugar-phosphate backbone near the polymerase active site is one parameter that influences the anti-syn equilibrium of 8-oxodG. Our results provide insights into the mechanisms employed by polymerases to select the complementary dNTP.     

Conformationally Restrained Chiral PNA Conjugates: Synthesis and DNA Complementation Studies

Abstract

In recent times, PNA (I), a structural mimic of DNA in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine (aeg) linkage has emerged as a potential antisense therapeutic agent.¹ A major limitation of PNAs from an application perspective is their poor solubility in aqueous medium and being achiral, they bind to cDNA in both parallel (N-PNA/5′-DNA) and antiparallel (N-PNA/3′-DNA) modes. In this connection, we have designed spermine conjugated and conformationally constrained PNA analogues to generate the 4-aminoprolyl backbone (II).² These were synthesised and evaluated for their DNA binding abilities by using UV and CD spectroscopic studies. It is seen that incorporation of one 4-aminoprolyl unit at the N-terminus of a PNA chain not only enhances the inherent binding of PNA to DNA, but also imparts significant bias in parallel and antiparallel binding with cDNA. Conjugation of spermine at C-terminus enhanced the PNA solubility.     

Mutagenesis, induced by phosphotriester analogs of oligonucleotides and directed to the cleavage site of double-spiral DNA]

The mutagenic properties of phosphotriester analogues revealed in course of interaction with linearized plasmid DNA were studied. The plasmid-based model system permitting one to test reliably the induced mutations is proposed. The efficiency of mutagenesis was shown to depend on the length of the oligonucleotide-mutagen and the genotype of the transformed Escherichia coli strain. The possible mechanisms involved in mutagenesis are discussed.     

The effect of electrostatic interactions on the conformation of the sugar-phosphate backbone in DNA]

The influence of sugar ring flexibility in DNA on the mechanism of the B<-->A conformational transition is studied. The dipole moment of the deoxyribose as a function of its puckered states is calculated by the quantum-mechanical method using the MINDO/3 approximation. The interaction of the sugar dipole with the neighbour molecular groups in polynucleotide chain is estimated. The sugar dipole interaction witch phosphate groups and counterions is shown to be strong and capable to deform the pseudorotation potential of deoxyribose. The effective pseudorotation potential of sugar ring in the B- and A-helices is obtained. The results are used to explain the behaviour of Raman bands in the region of sugar-phosphate vibrations. The mechanism of the effect of electrostatic forces on the sugar-phosphate backbone conformation which is essential for the B<-->A and other structure transitions is offered.     

Structural Aspects of Interaction of Homeodomains with DNA

This review is devoted to the structural aspects of interaction of homeodomains with DNA. Presented are the list of all homeodomains with known spatial structure and the alignment of their amino acid sequences. The structure of homeodomains and contacts of their amino acid residues with DNA bases and sugar-phosphate backbone are described. The role of water molecules in DNA binding is discussed. Structures of multicomponent protein complexes on DNA including homeodomains are characterized.     

Mutant Escherichia coli Ada proteins simultaneously defective in the repair of O6-methylguanine and in gene activation.

The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also possesses two distinct suicide alkyltransferase activities, for O6-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair O6-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal O6-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.     

Action models for the antitumor drug camptothecin: formation of alkali-labile complex with DNA and inhibition of human DNA topoisomerase I

The antitumor activity of camptothecin (CPT) and its derivatives, including water-soluble topotecan (TPT), is determined by their ability to inhibit human DNA topoisomerase I (top 1). On the other hand, TPT has been recently shown to bind to DNA. The proposed models are based on a two-step mechanism of TPT (CPT) dimer interaction with two spatially close DNA duplexes. At the first step, the CPT lactone form binds to DNA (Streltsov et al., Mol. Biol. vol. 36, no. 5 (2002)) through hydrogen bonding of its C16a carbonyl with the guanine 2-amino group. At the second step, CPT is converted to the carboxylate form. In the absence of top 1, the C17 hydroxyl of CPT is involved in ester exchange (nicking of the DNA sugar-phosphate backbone followed by covalent joining of free phosphate to C17) whereas its C20 carboxyl forms two hydrogen bonds with the same guanine nucleotide at the opposite end of the broken DNA backbone. As a result, CPT binds to both ends of the broken DNA. The resulting CPT-DNA complex is alkali-labile. In the presence of top 1, after CPT conversion to the carboxylate form and DNA nicking, the C17 hydroxyl makes a branching hydrogen bond with N1 and N3 of guanine while the C20 carboxyl makes two hydrogen bonds with the NH of Tyr723 and N(delta2)H(2) of Asp722. Owing to this, rotation of one end of the broken sugar-phosphate backbone about the other becomes impossible; hence the CPT inhibitory effect on top 1. The proposed models are consistent with the current body of experimental data.