ABSTRACT
Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.
Subject(s)
Copper/chemistry , DNA/chemistry , Netropsin/pharmacology , Oligopeptides/chemistry , Amino Acid Sequence , Base Sequence , Chelating Agents , Electrophoresis , Molecular Sequence Data , Netropsin/analogs & derivativesABSTRACT
An analog of the antibiotic netropsin containing two netropsin-like fragments linked covalently via a platinum atom has been synthesized. DNase I and hydroxyl radical footprinting studies have shown that this compound binds at selective sites on a DNA restriction fragment with a known nucleotide sequence. After X-ray irradiation of Pt-bis-netropsin--DNA complexes a platinum-mediated cleavage of DNA is observed at specific DNA sites. This enables one to determine the location of the synthetic ligand on the DNA with a precision of about one nucleotide. The cleavage activity seems to be related to the emission of Auger electrons from the platinum atom that cause rupture of the deoxyribose residues on the two DNA strands near the position of the platinum atom in the complex.
Subject(s)
DNA/genetics , Netropsin/analogs & derivatives , Organoplatinum Compounds/metabolism , Platinum/chemistry , Autoradiography , Base Sequence , DNA/chemistry , DNA/radiation effects , DNA Fingerprinting , Hydroxides , Hydroxyl Radical , Molecular Sequence Data , Netropsin/chemistry , Netropsin/metabolism , Nucleic Acid Conformation , Organoplatinum Compounds/chemistry , Plasmids , X-RaysSubject(s)
DNA Damage/radiation effects , DNA, Bacterial/radiation effects , DNA/radiation effects , Escherichia coli/genetics , Platinum/radiation effects , Base Sequence , Binding Sites/drug effects , Binding Sites/radiation effects , DNA/drug effects , DNA/genetics , DNA/metabolism , DNA Damage/genetics , DNA Damage/physiology , DNA, Bacterial/drug effects , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Glycerol/pharmacology , Ligands , Molecular Sequence Data , Netropsin/analogs & derivatives , Netropsin/metabolism , Netropsin/radiation effects , Plasmids/drug effects , Plasmids/genetics , Plasmids/radiation effects , SolutionsABSTRACT
Using spin trapping method there were discovered and identified the radicals of linolenic acid formed when initiating its peroxidation by the system Fe2+-ascorbate. Mechanism of formation of linolenic acid radicals and their role in initiation of peroxidation were studied. A scheme of reactions of peroxidation initiation in the system Fe2+-ascorbate. linolenic acid is proposed.
Subject(s)
Ascorbic Acid/metabolism , Ferrous Compounds/metabolism , Iron/metabolism , Linolenic Acids/metabolism , Lipid Peroxides/metabolism , Electron Spin Resonance Spectroscopy , Free Radicals , In Vitro Techniques , Kinetics , Oxidation-Reduction , alpha-Linolenic AcidABSTRACT
The mechanism of free radical generation in the reaction of ferrous ion with t-butyl and linolenic acid hydroperoxide was investigated by spin trapping method. The t-butoxyl, methyl, linolenic acid alkoxyl and alkyl radical spin adducts EPR spectra were observed and identified.
