ABSTRACT
Dissociation kinetics of triplexes formed by molecules of peptide nucleic acid (PNA) and DNA have been studied. The complexes consisted of oligomeric PNA containing 10 thymine bases and the dA(10) target incorporated in single-stranded (ssDNA) or double-stranded DNA (dsDNA). Their dissociation was followed by means of the gel mobility shift assay at various temperatures and sodium ion concentrations. In all experiments, the dissociation kinetics of triplexes were exponential; the effective lifetime of a triplex, tau, depended on temperature in accordance with the Arrhenius law. The tau values for T(10) PNA complexes with ss- and dsDNA were equal within the accuracy of experiments. The activation energy, U, value for T(10) PNA-DNA complexes did not change when the NaCl concentration was increased from 50 to 200 or 600 mM. Conversely, the tau values decreased with the increase in NaCl concentration. The equal lifetimes of the T(10) PNA-DNA triplexes containing ss- and dsDNA suggest that the loop formed in dsDNA does not noticeably affect the triplex structure. The decrease in the triplex lifetime tau with an increase in ionic strength was accounted for by the fact that the PNA backbone is neutral. The lack of relationship between the activation energy of dissociation and salt concentration suggests that the dissociation enthalpy does not depend on the ionic strength. Thus, the effect of ionic strength on the lifetime is entropic by its nature. Contrary to this, for complexes of ssDNA with bis-PNA 1743, which also consists of 10 thymine bases but contains 2 additional positive charges inside the sequence in 1 of the PNA arms, an increase of the dissociation enthalpy at low salt concentration was observed. We suggest that this effect is a result of a direct electrostatic interaction of the positive charges of the PNA with the DNA backbone. Finally, our results allow an estimate of the lifetime of a 10-mer triplex invasion complex in dsDNA at 37 degrees C in excess of several hundred days.
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Peptide Nucleic Acids/chemistry , Temperature , DNA, Single-Stranded/chemistry , Electrophoresis, Polyacrylamide Gel , Kinetics , Osmolar Concentration , Pyrimidines/chemistry , Sodium Chloride/chemistry , Spectrophotometry , ThermodynamicsABSTRACT
We studied modification of double-stranded DNA with chemically active homologous oligonucleotide derivatives carrying an alkylating 4-[N-2-chloroethyl-N-methylamino)benzyl]aminogroup in DNA recombination reaction promoted by E. coli RecA protein. It was shown that this chemical group does not prevent the formation of a complex between RecA protein and oligonucleotide as well as binding of this complex to double-stranded DNA. Formation of cross-links between DNA and oligonucleotide derivatives was observed at oligonucleotide lengths of no less than 30. The supercoiled form of the plasmid was alkylated with a higher efficacy than relaxed or linear form. In spite of homology between oligonucleotide and a certain region of double-stranded DNA (pBR322), cross-links were observed throughout the DNA length including nonhomologous regions. The causes of such behavior are discussed.
Subject(s)
DNA, Recombinant/metabolism , Escherichia coli/metabolism , Oligonucleotides/metabolism , Rec A Recombinases/metabolism , Alkylation , Cross-Linking ReagentsABSTRACT
The distribution of guanine-cytosine (GC) pairs in the DNA of the highly oncogenic simian adenovirus type 7 (SA7) and the non-oncogenic human adenovirus type 6 (Ad6) has been studied by thermal denaturation and CsC1 density-gradient centrifugation. The differential of the DNA thermal denaturation curves shows the presence of pronounced peaks which indicates uneven distribution of GC pairs along the DNA chains and the presence of regions with GC content from 30 to 74% in SA7 DNA and from 40 to 68% in Ad6 DNA. The DNA restriction fragments obtained by treatment with EcoRI, BamHI, SalI, BglII and HindIII were subjected to CsC1 density-gradient centrifugation. GC content of the fragments ranged from 45 to 70% for SA7 DNA and from 43 to 61% for Ad6 DNA. The GC content of the extreme left-hand fragments, where the transforming gene(s) is located, was higher than the average for SA7 DNA and lower than the average for Ad6 DNA. The most GC-rich regions were localized in the centre of the genome. The GC content of the right-hand part of both viral genomes was lower than the average.
