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PubMedDate not supplied

Synthesis and structural characterization of the N2G-mitomycin C-N2G interstrand cross-link in a model synthetic 23 base pair oligonucleotide DNA duplex.

Mitomycin C (MMC) is a genotoxic cancer chemotherapeutic agent that reacts principally at the N2 position of guanine to form one of two predominant monoadducts, or a G-G interstrand cross-link at CpG sites, or a G-G intrastrand cross-link at GpG sites. Previous studies of MMC adduction have principally used very short duplex oligonucleotides (5-15 bp) or very long native duplex DNAs. We examined the formation and structural features of the MMC CpG interstrand cross-link on a model 23 bp synthetic oligonucleotide duplex having the (upper strand) sequence 5'-ATAAATACGTATTTATTTATAAA-3'. MMC was reacted with the duplex oligonucleotide in the presence of sodium dithionite at ratios of 6 mM dithionite: 1.5 mM MMC:0.03 mM duplex. The yield of cross-link in the reaction was determined to be approximately 4.8% by denaturing gel electrophoresis, which represented approximately 75% of the total bound MMC. The cross-linked DNA was isolated to greater than 97% purity in a single step by high temperature size exclusion column chromatography. Characterization of the purified product confirmed that the complex contained exclusively the N2G-MMC-N2G cross-link at the single central CpG site. CD spectroscopy demonstrated a negative band at approximately 290-320 nm which has previously been shown to be characteristic of the MMC cross-link. The relative intensity of this band compared to those reported for shorter duplexes suggested that the majority of the duplex is in a normal B-DNA helical configuration. Base-specific chemical footprinting techniques also indicated that there were subtle but distinct structural perturbations principally within the central four to six base pairs containing and adjacent to the cross-link.

Base Composition↗

PubMed2006

Effects of the modified aromatic ring of WNA on stability of triplex DNA.

Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but their recognizable duplexes are limited to homopurine/homopyrimidine sequences because of interruption of pyrimidine base in the purine strand. This problem has not been fully solved despite numerous studies. We have previously reported that the novel nucleoside analogues (WNA: W-shaped nucleoside analogues), WNA-betaT and WNA-betaC, can recognize a TA and a CG interrupting sites to form triplexes with high stability and selectively, respectively. However, further investigations have shown that the triplex formation using the WNA derivatives is dependent on its neighbouring bases of the TFOs. In this study, the new WNA analogues having a variety aromatic ring were synthesized to evaluate effects on sequence dependency. And it was found that changes in the substituent of aromatic ring of WNA derivatives altered the binding selectivity and stability in the triplex formation.

DNA↗

PubMed1999

Crystal structure of a cobalt-activated diphtheria toxin repressor-DNA complex reveals a metal-binding SH3-like domain.

The diphtheria toxin repressor (DtxR) is the prototype of a family of iron-dependent regulator (IdeR) proteins, which are activated by divalent iron and bind DNA to prevent the transcription of downstream genes. In Corynebacterium diphtheriae, DtxR regulates not only the expression of diphtheria toxin encoded by a corynebacteriophage, but also of components of the siderophore-mediated iron-transport system. Here we report the crystal structure of wild-type DtxR, a 226 residue three-domain dimeric protein, activated by cobalt and bound to a 21 bp DNA duplex based on the consensus operator sequence. Two DtxR dimers surround the DNA duplex which is distorted compared to canonical B -DNA. The SH3-like third domain interacts with the metal at site 1 via the side-chains of Glu170 and Gln173, revealing for the first time a metal-binding function for this class of domains. The SH3-like domain is also in contact with the DNA-binding first domain and with the second, or dimerization, domain. The DNA-binding helices in the first domain are shifted by 3 to 5 A when compared to the apo-repressor, and fit into the major groove of the duplex bound. These shifts are due to a hinge-binding motion of the DNA-binding domain with respect to the dimerization domains of DtxR. The third domain might play a role in regulating this hinge motion.

Bacterial Proteins↗

PubMed1997

Structural analysis of DNA bending induced by tethered triple helix forming oligonucleotides.

In order to monitor DNA flexibility, we have recently reported the design of an artificial DNA bending system consisting of two triple helix forming oligonucleotides (TFOs) connected by a flexible linker [Akiyama, T., & Hogan, M. E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12122-12127], which spans a single turn of DNA helix. Those data suggested that up to 60 degrees of bending deformation could be induced with an expenditure of energy which is much smaller than predicted from bulk flexibility parameters. In this report, the detailed structure of the bend has been investigated by three different methods: circular permutation analysis, phasing analysis, and ring closure. Circular permutation and phasing analysis suggest that the magnitude of the bend is dependent on linker length. The apparent location of the bend was estimated from circular permutation analysis to be at the duplex region intervening the two sites of triple helix formation. The electrophoretic mobility of the bent complex appears to vary with the sequence of the intervening duplex region of the binding site complex, in the order of AT-rich > random > or = GC-rich sequence. Detailed fitting of the phasing data has shown that bending is not accompanied by significant twisting deformation. Ring closure analysis with T4 DNA ligase has confirmed the general magnitude of the TFO-induced bend and has additionally suggested that formation of the simple linear antiparallel triple helix does not enhance DNA flexibility.

DNA↗

PubMed1987

[Chemical synthesis and properties of oligonucleotide substrates for restriction endonuclease BamHI and methyltransferase Eco dam].

Oligodeoxyribonucleotides which form a number of duplexes, containing the recognition sequences for endonuclease BamHI and DNA methylase Eco dam, were synthesised by the phosphotriester approach. Furthermore, synthesis of 3'-phosphorylated oligodeoxyribonucleotides from corresponding S-methyl phosphorothioate triester oligomers is described. The synthetic duplexes are characterized by some defects in the recognition sequences for endonuclease BamHI and methylase Eco dam, viz. nick, absence of an internucleotide phosphate, modifications (including partial single-strandedness) of the recognition site. Interaction of the enzymes with these synthetic substrates was investigated.

Base Sequence↗

PubMed1984

Effect of environment, conformation, sequence and base substituents on the imino proton exchange rates in guanine and inosine-containing DNA, RNA, and DNA-RNA duplexes.

High-resolution 1H nuclear magnetic resonance in H2O has been used to study the effect of sequence, conformation, environmental factors and base substituents on the exchange behavior of the hydrogen-bonded imino protons of guainine X cytosine and inosine X cytosine base-pairs in DNA, RNA, and DNA-RNA duplexes. The exchange rates were determined by measurement of the spin-lattice relaxation rates of the imino protons as a function of temperature. The exchange was not altered by the presence of high concentrations of salt, and the inability of phosphate to catalyze the exchange indicates that the exchange is limited by formation of a solvent-accessible "open" state. The exchange behavior depends on the duplex conformation and sequence. Exchange from the Z form polymers was orders of magnitude slower than the corresponding duplexes in the B conformation, and the A form RNA duplexes exchanged more slowly than the B form DNA polymers with the same sequence. The exchange behavior of the DNA-RNA hybrids was dependent on whether the purine or the pyrimidine strand contained the deoxyribose sugar. For both the guanine and inosine-containing duplexes, the homopolymer duplexes exchange more slowly than the more stable alternating copolymers. For the alternating duplexes, substitution of cytosine with 5-bromo- or 5-methylcytosine slowed the exchange and increased the activation energy for exchange. The inosine-containing duplexes exchanged more rapidly than the guanosine-containing duplexes, but both showed similar changes in exchange behavior in response to changes in sequence and base substituents. The activation energies for base-pair opening in B form DNA are correlated with the van der Waals contribution to the base-base interaction energy, suggesting that the purine base is partially unstacked in the open state. Using the relaxation measurements to set an upper limit on the exchange rate in poly(dG-dC) and the tritium exchange behavior at low temperature, we find that even though Z-DNA exchanges very slowly, the activation energy is similar to that observed in the A and B form duplexes, suggesting that exchange occurs from a similar open state.

Base Composition↗

PubMed2006

Overview of the structure of all-AT oligonucleotides: organization in helices and packing interactions.

We present the crystalline organization of 33 all-AT deoxyoligonucleotide duplexes, studied by x-ray diffraction. Most of them have very similar structures, with Watson-Crick basepairs and a standard average twist close to 36 degrees. The molecules are organized as parallel columns of stacked duplexes in a helical arrangement. Such organization of duplexes is very regular and repetitive: all sequences show the same pattern. It is mainly determined by the stacking of the terminal basepairs, so that the twist in the virtual TA base step between neighbor duplexes is always negative, approximately -22 degrees. The distance between the axes of parallel columns is practically identical in all cases, approximately 26 A. Interestingly, it coincides with that found in DNA viruses and fibers in their hexagonal phase. It appears to be a characteristic distance for ordered parallel DNA molecules. This feature is due to the absence of short range intermolecular forces, which are usually due to the presence of CG basepairs at the end of the oligonucleotide sequence. The duplexes apparently interact only through their diffuse ionic atmospheres. The results obtained can thus be considered as intermediate between liquid crystals, fibers, and standard crystal structures. They provide new information on medium range DNA-DNA interactions.

Base Composition↗

PubMed1994

Crystal structure of the highly distorted chimeric decamer r(C)d(CGGCGCCG)r(G).spermine complex--spermine binding to phosphate only and minor groove tertiary base-pairing.

The crystal structure of the self-complementary chimeric decamer duplex r(C)d(CGGCGCCG)r(G), with RNA base pairs at both termini, has been solved at 1.9 A resolution by the molecular replacement method and refined to an R value of 0.145 for 2,314 reflections. The C3'-endo sugar puckers of the terminal riboses apparently drive the entire chimeric duplex into an A-DNA conformation, in contrast to the B-DNA conformation adopted by the all-deoxy decamer of the same sequence. Five symmetry related duplexes encapsulate a spermine molecule which interacts with ten phosphate groups, both directly and through water molecules to form multiple ionic and hydrogen bonding interactions. The spermine interaction severely bends the duplexes by 31 degrees into the major groove at the fourth base pair G(4).C(17), jolts it and slides the 'base plate' into the minor groove. This base pair, together with the adjacent base pair in the top half and the corresponding pseudo two-fold related base pairs in the bottom half, form four minor groove base-paired multiples with the terminal base pairs of two neighboring duplexes.

Base Composition↗

PubMed1993

Oligodeoxynucleotides containing 2'-O-modified adenosine: synthesis and effects on stability of DNA:RNA duplexes.

Hybridization thermodynamics were compared for oligonucleotide sequences containing 2'-fluoro dA, 2'-O-methyl A, 2'-O-ethyl A, 2'-O-propyl A, 2'-O-butyl A, 2'-O-pentyl A, 2'-O-nonyl A, 2'-O-allyl A, and 2'-O-benzyl A in place of deoxyadenosine. Although the effect of 2'-modified adenosine on duplex stability is sequence dependent, a clear trend is apparent. For six sequences containing a few 2'-modified adenosines in a background of unmodified deoxynucleotides, the average delta TM per substitution ranged from +1.3 degrees C for 2'-fluoro dA to -2.0 degrees C for 2'-O-nonyl A. For the 2'-O-alkyl series, the average delta TM per substitution correlates well with size of the substituent; the order of stability is 2'-O-methyl A > 2'-O-ethyl A > 2'-O-propyl A > 2'-O-butyl A > 2'-O-pentyl A > 2'-O-nonyl A. This correlation also extends to 2'-fluoro dA, 2'-O-allyl A, and 2'-O-benzyl A if chain length is measured by number of carbon atoms. When examined in the background of 2'-O-methyl ribonucleotides, all 2'-modified adenosines with a substituent no larger than 2'-O-pentyl stabilized the duplex nearly 2 degrees C per substitution compared to unmodified dA. These thermodynamic results and CD spectra of modified and unmodified hybrids support a model of DNA:RNA hybrids in which the geometry is between that of B-form and A-form.

Adenosine↗

PubMed1999

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.

Base Sequence↗

PubMed2003

Enthalpy/entropy compensation: influence of DNA flanking sequence on the binding of 7-amino actinomycin D to its primary binding site in short DNA duplexes.

The effect of the context of the flanking sequence on ligand binding to DNA oligonucleotides that contain consensus binding sites was investigated for the binding of the intercalator 7-amino actinomycin D. Seven self-complementary DNA oligomers each containing a centrally located primary binding site, 5'-A-G-C-T-3', flanked on either side by the sequences (AT)(n) or (AA)(n) (with n = 2, 3, 4) and AA(AT)(2), were studied. For different flanking sequences, (AA)(n)-series or (AT)(n)-series, differential fluorescence enhancements of the ligand due to binding were observed. Thermodynamic studies indicated that the flanking sequences not only affected DNA stability and secondary structure but also modulated ligand binding to the primary binding site. The magnitude of the ligand binding affinity to the primary site was inversely related to the sequence dependent stability. The enthalpy of ligand binding was directly measured by isothermal titration calorimetry, and this made it possible to parse the binding free energy into its energetic and entropic terms. Our results reveal a pronounced enthalpy-entropy compensation for 7-amino actinomycin D binding to this family of oligonucleotides and suggest that the DNA sequences flanking the primary binding site can strongly influence ligand recognition of specific sites on target DNA molecules.

Antineoplastic Agents↗

PubMed1995

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.

Animals↗

PubMed1994

Genetic recombination of nucleosomal templates is mediated by transcription.

An in vitro system has been developed to examine the influence of transcription on genetic rearrangement. Using a homologous pairing assay, the transfer of one strand of a nucleosomal template onto a recipient DNA molecule was monitored as a function of RNA polymerase activity. Transcriptionally inactive nucleosomal DNA was refractory to homologous pairing. Homologous pairing was catalyzed, however, by the eukaryotic recombinase, rec1, when the nucleosomal template was being transcribed. The reaction was found to be dependent on the presence of rec1, RNA polymerase, NTPs and RNA synthesis. Heteroduplex formation between a short DNA duplex fragment assembled into a nucleosome and a single-stranded circle relied also on the presence of sequence homology between the duplex and the circle. The results of this study lend support to the notion that transcriptionally active regions within a chromosome are more apt to serve as sites of genetic recombination.

Cell-Free System↗

PubMed1994

Properties of multiple G.A mismatches in stable oligonucleotide duplexes.

The solution structure of the deoxydecanucleotide [sequence: see text] has been determined by NMR methods. This duplex, which contains six G.A mismatches and four Watson-Crick base pairs, is thermodynamically more stable than a decamer where T.A base pairs are substituted for the G.A mismatches, and is less stable than the duplex that contains G.C base pairs. Circular-dichroism spectroscopy indicates an overall B-like conformation for the decamer, but stronger than usual base stacking. 1H-NMR spectroscopy revealed that the N1H groups of the mismatched guanine residues are not hydrogen bonded, and 31P-NMR showed the presence of BII phosphate conformations for the GpA steps. Detailed analysis of the NMR data showed that all nucleotides have anti glycosidic torsion angles and S type sugar puckers. The G.A mismatches pair in the amino form as originally proposed by Li et al. [Li, Y., Zon, G. & Wilson, W. D. (1991) Proc. Natl Acad. Sci. USA 88, 26-30], which results in extensive base-base stacking between the tandem G.A base pairs and their nearest neighbours. The terminal G.A base pairs are less stable than the central base pairs and show evidence of an equilibrium between two conformations, one involving BII phosphate.

Base Sequence↗

PubMed2006

Synthesis and characterization of enantiomeric anti-2-fluorobenzo[a]pyrene-7,8-dihydrodiol-9,10-epoxides and their 2'-deoxyguanosine and oligodeoxynucleotide adducts.

Benzo[a]pyrene diol epoxides (BPDEs) are the ultimate carcinogenic species of benzo[a]pyrene, a prototype polycyclic aromatic hydrocarbon (PAH). BPDE-modified DNA duplexes can adopt multiple conformations depending on the nature of the modified bases, the stereochemistry at the location of the covalent linkage, and the sequence context surrounding the lesion site. In this paper, we describe the preparation of enantiomeric 2-fluoro-BPDEs, trans-(7R,8S)-dihydroxy-(9S,10R)- and trans-(7S,8R)-(9R,10S)-epoxy-7,8,9,10-tetrtahydro-2-fluorobenzo[a]pyrene (22 and 23, respectively), as models for probing the BPDE-induced conformational heterogeneity. The multistep synthesis of the target diol epoxides described herein entails regiospecific succinoylation of 2-fluoropyrene, followed by a ring closure, regio- and stereospecific construction of the 7,8-dihydrodiol functionality, and a subsequent meta-chloroperbenzoic acid-mediated epoxidation. Stereoselectivity was achieved by using Jacobsen chiral catalysts, which produced greater than approximately 90% enantiomeric excess. Absolute configurations at the C(7,8) carbons of the FBP derivatives were determined by comparison of the circular dichroism (CD) spectra with those reported for the BP analogues. Analysis of the 3J(7,8) vicinal coupling constants, CD shape, and charge density calculations all indicated that the prepared anti-FBPDEs preferentially adopt the pseudo-diequatorial C(7,8) diol conformation. Hydrolysis of anti-FBPDEs produced a 9:1 ratio of trans- to cis-opened tetraols. Reactions of each of the anti-FBPDEs with deoxyguanosine 5'-monophosphate produced predominantly trans-anti-N2-dG as the major adducts. Analogous reactions with two 11-mer oligodeoxynucleotides (5'-CCATXGCTACC-3' where X = dT, dC) gave FBP-modified oligodeoxynucleotides with structures that were characterized by enzyme digest/HPLC and electrospray ionization time-of-flight mass spectrometry data. The oligonucleotide adducts were annealed with the appropriate sequences to form fully complementary duplexes [(5'-CCATXGCTACC-3')(5'-GGTAGCYATGG-3'), G = FBP-N2-dG adduct, X = dT, Y = dA in duplex I; X = dC, Y = dG in duplex II] for CD and UV melting studies. The results of the present study were consistent with those reported previously for BPDE-modified duplexes in the same sequence contexts and support the utility of FBPDEs as useful structural probes.

DNA Adducts↗

PubMed1998

A PCR-based method for uniform 13C/15N labeling of long DNA oligomers.

A polymerase chain reaction (PCR)-based method is described for uniform 13C/15N labeling of DNA duplexes. In this method, multiple copies of a blunt-ended duplex are cloned into a plasmid with each copy containing the sequence of interest and the restriction HincII sequences at the 5' and 3' ends. PCR with uniformly 13C/15N-labeled dNTP precursors results in a labeled DNA duplex containing multiple copies of the sequence of interest. Use of bi-directional primers, instead of self-priming [Louis et al. (1998) J. Biol. Chem. 273, 2374-2378], produces a DNA fragment of unique length. Twenty-four cycles of PCR of this purified product followed by restriction and purification gives (with 30% yield) the uniformly 13C/15N-labeled duplex sequence for multi-nuclear magnetic resonance spectroscopy.

Base Sequence↗

PubMed1990

Characterization of the structure and melting of DNAs containing backbone nicks and gaps.

A DNA molecule containing a gap (a missing phosphate) has been examined and compared to two other molecules of the same sequence, one containing a nick (a phosphorylated gap) and the other a normal duplex containing no break in the backbone. A second gapped sequence was also compared to a normal duplex of the same sequence. The molecules containing nicks or gaps were generated as dumbbell molecules, short helices closed by a loop at each end. The dumbbells were formed by the association of two hairpins with self-complementary dangling 5'-ends. Nuclear magnetic resonance was used to monitor the melting transition and to probe structural differences between molecules. Under the conditions used here no change in stability was observed upon phosphorylation of the gap. Structural changes upon phosphorylation of a gap or closure of a nick were minimal and were localized to the region immediately around the gap or nick. Two transitions can be observed as a gapped or nicked molecule melts, although the resolution of the two transitions varies with the salt concentration. At moderate to high salt (greater than or equal to 30 mM) the molecule melts essentially all at once. At low salt the two transitions occur at temperatures that differ by as much as 15 degrees C. In addition, comparison with other NMR melting studies indicates that the duplex formed by the overlap of the dangling ends of the hairpins is stabilized relative to a free duplex of the same sequence, probably by stacking onto the hairpin stem.

Base Composition↗

PubMed1986

"In vitro" method of assembling a synthetic gene.

Without prior in vitro enzymatic ligation a DNA duplex was assembled successfully by directly transforming competent cells with a mixture containing six synthetic complimentary oligodeoxyribonucleotides and a linearized plasmid. One out of 100 transformants was positive in colony hybridization with one of the synthetic fragment probe. The sequence of the DNA duplex inserted into the plasmid was confirmed by dideoxy sequencing method.

Base Sequence↗