SPECIFIC DNA CLEAVAGE BY A NETROPSIN ANALOG CONTAINING A COPPER(II)-CHELATING PEPTIDE GLY-GLY-HIS

Experimental data are presented for the ability of synthetic netropsin analogs to cleave the DNA sugar-phosphate backbone under certain cond itions. The netropsin analogs consist of two N-propylpyrrole carboxami de fragments covalently linked to a Cu2+-chelating tripeptide-glycyl-g lycyl-L-histidine-through two or three glycine residues. Incubation of a DNA restriction fragment and the netropsin analog in the presence o f sodium ascorbate, hydrogen peroxide, and Cu2+ results in DNA cleavag e at the sites of preferred binding of the netropsin analog. Similar c leavage is observed after X-ray irradiation of DNA complexes with the copper(II)-containing netropsin analog. The pattern and the intensity of DNA cleavage are dependent on the length of the chain connecting th e histidine-containing peptide with the N-propylpyrrole carboxamide fr agments. The netropsin analog containing three glycine residues in the connecting chain, in contrast to the analog with a shorter connecting chain, is capable of efficiently cleaving one of two polynucleotide s trands at the site of presumed binding of the dimer form of the analog . After X-ray irradiation more than 50% of total DNA can be cleaved at this position. Analysis of the nucleotide sequence adjacent to the si tes of preferred cleavage in several DNA restriction fragments shows t hat the consensus is 5'-TTTTNCAAAA-3', where N is an arbitrary nucleot ide. Copper(II)-mediated DNA cleavage takes place at the second adenin e from the 5'-end (marked by asterisk). The most efficient cleavage is observed at a molar ratio of Cu2+ to the netropsin analog equal to 0. 5. The free and the Cu2+-carrying oligopeptide appear to interact with each other to form a heterodimer which binds to the DNA at the cleava ge site. To verify this model, the binding of the free netropsin analo g and its Cu2+ chelate with a self-complementary oligonucleotide 5'-GC GTTTTGCAAAACGC-3' was studied. The binding of chelate to oligonucleoti de preincubated with the netropsin analog in the absence of Cu2+ was s hown to be a cooperative process stipulated by the interaction between two bound ligands. The cooperativity parameter was estimated at appro ximately 6. A model is proposed according to which the heterodimer sta bilized by an interligand beta-sheet is incorporated into the DNA mino r groove.