Electron Microscopic and Physico-Chemical Studies of DNA Complexes with Synthetic Oligopeptides: Binding Specificity and DNA Compact Structures

Abstract

Binding to DNA of two synthetic peptides, Val-Thr-Thr-Val-Val-NH-NH-Dns and Thr-Val- Thr-Lys-Val-Gly-Thr-Lsy-Val-Gly-Thr-Val-Val-NH-NH-Dns (where Dns is a residue of 5- dimethylaminonaphthalene-l-sulfonic acid), has been studied by circular dichroism, electron microscopy and fluorescence methods. It has been found that these two peptides can self- associate in aqueous solution as follows from the fact that concentration-dependent changes are observed in the UV absorbance and fluorescence spectra. The two peptides can bind to DNA both in self-associated and monomeric forms. The pentapeptide in the β-associated form binds more strongly to poly(dG) · poly(dC) than to poly[d(A-C)] · poly[d(G-T)] and poly(dA) · poly(dT) whereas the tridecapeptide exhibits an opposite order of preferences binding more strongly to poly[d(A-C)] · poly[d(G-T)] and poly(dA) · poly(dT) than to poly(dG) · poly(dC).

Binding is a cooperative process which is accompanied by the DNA compaction at peptide/DNA base pair ratios greater than l. At the initial stage of the compaction process, the coalescence of DNA segments covered by bound peptide molecules leads to the formation of DNA loops stabilized by the interaction between peptide molecules bound to different DNA segments. Further increase in the peptide/DNA ratio leads to the formation of rod-like structures each consisting of two or more double-stranded DNA segments. The final stage of the compaction process involves folding of fibrillar macromolecular complexes into a globular structure containing only one DNA molecule.     

The Specificity of Antibodies Elicited by Poly(dG)-Poly(dC)

Abstract

Antibodies have been raised to the synthetic DNA polymer poly(dG)·poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.     

Synthesis of an Enantiomerically Pure Carbocyclic DNA Abasic Site Analogue

Abstract

A short synthetic route to an appropriately derivatized carbocyclic analogue of abasic site residues of DNA is proposed.     

A comparative study of genomes in angiosperms

Angiosperms investigated by DNA/DNA reassociation studies were classified and tested for a taxonomic class- and subclass-specifity in a biometrical fashion. Monocotyledons and Dicotyledons differ significantly from each other with respect to a genomic parameter (U/R-ratio;U single copy DNA fraction;R = 1-U fraction of repetitive DNA). This difference is discussed from an evolutionary and molecular point of view.—Intercorrelations between the fraction of fast repeats, slow repeats, and single copy DNA can be detected. The amount of DNA organized in a short period pattern of interspersion is found to depend on the fraction of repetitive and single copy DNA. The number of DNA segments tandemly arranged in a short period pattern is linearly correlated withR/U-values. This correlation allows for a formula suitable for the estimation of the number of active genes in angiosperms. The analytical complexities of repetitive and single copy DNA are linearly correlated with the genome size of higher plants. The ratioU/R depends on the genome size of angiosperms in a hyperbolic fashion.     

A one-step method for quantitative determination of uracil in DNA by real-time PCR

Uracil may occur in DNA due to either cytosine deamination or thymine replacing incorporation. Its quantitative characterization is important in assessing DNA damages in cells with perturbed thymidylate metabolism or within different DNA segments involved in immunoglobulin gene diversification. The archaeal DNA polymerase from Pyrococcus furiosus binds strongly to the deaminated base uracil and stalls on uracil-containing templates. Here, we present a straightforward method for quantitative assessment of uracil in DNA within specific genomic segments. We use wild-type P. furiosus polymerase in parallel with its point mutant version which lacks the uracil-binding specificity on synthetic and genomic DNA samples to quantify the uracil content in a single-step real-time PCR assay. Quantification of the PCR results is based on an approach analogous to template copy number determination in comparing different samples. Data obtained on synthetic uracil-containing templates are verified by direct isotopic measurements. The method is also tested on physiological DNA samples from Escherichia coli and mouse cell lines with perturbed thymidylate biosynthesis. The present PCR-based method is easy to use and measures the uracil content within a genomic segment defined by the primers. Using distinct sets of primers, the method allows the analysis of heterogeneity of uracil distribution within the genome.     

Nucleoside Phosphonates. Development of Synthetic Methods and Reagents

Abstract

In this paper a short account of our recent research concerning development of new synthetic methods and new reagents for the preparation of DNA and RNA fragments and their analogues is given.

     

Construction of Genes or Gene Fragments by Use of Two Long Synthetic Oligonucleotides Representing the Coding and Noncoding Strands

Abstract

The construction of genes as exemplified for a gene encoding salmon calcitonin-gly(33) from only two long synthetic oligonucleotides (coding/noncoding strand) is described. The type of solid support used in oligonucleotide preparation strongly influences synthesis yields and mutation rates in the cloning of the synthetic DNA.     

Enhancement of Stability of Double and Triple Stranded DNA by Cationic Amphiphilic α-Helix Peptide

Abstract

It was found that synthetic amphiphilic α-helix peptide could bind stabilize double or triple stranded DNA. The stabilization effect was significant for cationic α-helix peptides which indicated the importance of electrostatic interaction of positive charge of peptide and negative charge of DNA. It was also pointed out that α-helix content was increased in the presence of DNA.     

Synthesis of Oligonucleotides Containing the Abasic Site Model Compound 1,4-Anhydro-2-Deoxy-D-Ribitol

Abstract

An improved approach for the synthesis of phosphotriester and phosphoramidite derivatives of the 1,4-anhydro-2-deoxy-D-ribitol is presented. The incorporation of these compounds in synthetic DNA and the insertion of purine deoxyribonucleotides opposite the reduced abasic site by DNA polymerase is described.     

B-Z Junctions in Supercoiled pRW751 DNA Contain Unpaired Bases or Non-Watson-Crick Base Pairs

Abstract

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)₁₃ and (dC-dG)₁₆ segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA but only the latter requires non-paired bases for the reaction. Nuclease SI and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the “outer” boundaries between the (dC-dG)_n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751.

As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)_n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease SI sensitive sites. The results suggest that the “outer” B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson- Crick base pairs.     

Recognition of the Structural Distortions at the Junctions Between B and Z Segments in Negatively Supercoiled DNA by Osmium Tetroxide

Abstract

It has been shown for the first time that conformational junction between contiguous right- handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)₁₃ and (dC-dG)₁₆ blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease SI cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)_n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)₁₃ segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)_n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.     

Mutation Induced by Deoxyxanthosine in Codon 12 of A Synthetic c-Ha-ras Gene

Abstract

To evaluate the base-pairing properties and mutagenicity of deoxyxanthosine in DNA, the modified base was incorporated into a synthetic c-Ha-ras gene and a DNA transfection experiment was done. The ras gene containing deoxyxanthosine showed very high focus-forming activity. Analysis of the genes from transformants showed almost exclusively a transition of G to A. These results indicate that dTMP was preferentially incorporated at the site opposite to deoxyxanthosine, and deoxyxanthosine can induce G to A transitions in mammalian cells.     

Synthesis of Duplex DNA Containing a Spin Labeled Analog of 2′ Deoxycytidine

Abstract

We report the chemical synthesis of phosphoramidite 8, containing a spin labeled analog of deoxycytidine, C?, and its incorporation into synthetic DNA. The EPR characteristics of the resulting DNAs indicated that the motion of the spin label was well-correlated with the uniform modes of the macromolecule, but that correlation of the spin label with internal motion was less effective than that achieved using a spin labeled quinolone, Q.     

A New Type of DNA Polymorphism Identified in the Species-Specific DNA Region Originating from the <Emphasis Type="Italic">Candida albicans</Emphasis> Mitochondrial Genome

Mitochondrial DNA of the pathogenic yeast Candida albicans contains a species-specific DNA region, designated EO3, within the duplicated region in its stem-and-loop structure, as reported in our previous paper. We have revealed that the EO3 region exhibits a DNA-size polymorphism, yielding three fragment length types: L, M, and S. This polymorphism is due to the presence (or absence) of two small nucleotide sequences of ~50 bp (designated a and b) within the EO3 region: type L possesses both a and b, type M possesses a alone, and type S possesses neither a nor b. Here we have identified a new type of EO3, which possesses b but not a, designated type M-II. Restriction fragment analyses with BglII revealed accurate discrimination of EO3 into four types: L, M-I, M-II, and S. Moreover, we show that these small nucleotide segments a and b were encompassed by the characteristic nucleotide sequence with a common inverted repeat structure. In the present report we propose a revised version of EO3 typing, suggesting epidemiological usefulness as an alternative tool for species-specific detection of C. albicans, and discuss the potentiality of EO3 for generating more variations of DNA polymorphism.     

Novel 1-acyl-4-substituted semicarbazide derivatives of primaquine − synthesis,cytostatic, antiviral and antioxidative studies

A series of novel 1,4-substituted semicarbazides 5a–g with a primaquine moiety bridged by a carbonyl group at position 1 and a cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 were prepared and biologically evaluated. The synthetic pathways applied for preparation of the title compounds involved benzotriazole as synthetic auxiliary. Primaquine semicarbazides 5a–g and their synthetic precursors benzotriazolecarbonyl semicarbazides 4 were evaluated for cytostatic, antiviral and antioxidative activities. All compounds of the series 5 showed high selectivity towards MCF-7 cells (breast carcinoma) with IC₅₀ values in the low micromolar range and the most active was benzyl derivative 5c (IC₅₀ 1?±?0.2 µM). The benzhydryl derivative 5e showed significant cytostatic activities towards all the tested cell lines (IC₅₀ 4–18 µM). The same compound was the strongest lipoxygenase inhibitor as well (51%). The highest antioxidant activity was demonstrated for the hydroxy derivative 5g and benzotriazolecarbonyl semicarbazides 4b,c (61.2–68.5%). No antiviral activity was observed against a wide variety of DNA and RNA viruses.     

Osmotic Pressure of DNA Solutions and Effective Diameter of the Double Helix

Abstract

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col El plasmid DNA solutions in the concentration range of 1–4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A ₂ of the solution of DNA segments and the effective chain diameter d _eff in the ionic strength range of 10^?2-0.1 M, As the ionic strength is lowered from 0.1 to 10^?2 M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.     

Binding of Bis-linked Netropsin Derivatives in the Parallel-Stranded Hairpin Form to DNA

Abstract

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5′-CCTATATCC-3′ in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis -diammine Pt(II)- bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5′-CCTATATCC-3′ (I), 5′-CCTTAATCC-3′ (II), 5′-CCTTATTCC-3′ (III), 5′-CCTTTTTCC-3′ (IV) and 5′-CCAATTTCC-3′ (V) decreases in the order I = II > III > IV> V. The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.     

Alien DNA introgression and wheat DNA rearrangements in a stable wheat line derived from the early generation of distant hybridization

Polyploidy has been found to be common in plants. Bread or common wheat (Triticum aestivum L., 2n=42) is a good example of allopolyploid made up of three diploid genomes A, B and D. In recent years, by the study of mimicking the origination of common wheat, it was found that changes of DNA sequence and gene expression occurred at the early stages of artificial allohexaploid between tetraploid wheat and Aegilops tauschii, which was probably favorable to genetic diploidization of new synthetic hexaploid wheat. Common wheat 99L2 is a new line stable in genetic, which was derived from the early self-pollinated generation of wide hybrids between common wheat and rye. In this study, it was found that at least two rye DNA segments had been introgressed into 99L2. This result suggested that a mechanism of alien DNA introgression may exist, which was different from the traditional mechanism of chromosome pairing and DNA recombination between wheat and alien species. Meanwhile, during the introgression process of alien rye DNA segments, the changes in DNA sequences of wheat itself occurred.     

Left-handed DNA Crossovers. Implications for DNA-DNA Recognition and Structural Alterations

Abstract

The close approach of DNA segments participates in many biological functions including DNA condensation and DNA processing. Previous crystallographic studies have shown that B-DNA self-fitting by mutual groove-backbone interaction produces right-handed DNA crossovers. These structures have opened new perspectives on the role of close DNA-DNA interactions in the architecture and activity the DNA molecule. In the present study, the analysis of the crystal packing of two B-DNA decamer duplexes d(CCIIICCCGG) and d(CCGCCGGCGG) reveals the existence of new modes of DNA crossing. Symmetric left- handed crossovers are produced by mutual fitting of DNA grooves at the crossing point. New sequence patterns contribute to stabilize longitudinal fitting of the sugar-phosphate backbone into the major groove. In addition, the close approach of DNA segments greatly influences the DNA conformation in a sequence dependent manner. This study provides new insights into the role of DNA sequence and structure in DNA-DNA recognition. In providing detailed molecular views of DNA crossovers of opposite chirality, this study can also help to elucidate the role of symmetry and chirality in the recognition of complex DNA structures by protein dimers or tetramers, such as topoisomerase II and recombinase enzymes. These results are discussed in the context of the possible relationships between DNA condensation and DNA processing.