366 - Polarographie Behaviour of Double-Helical DNA Containing Intramolecular Single Stranded Regions

Abstract

Calf thymus DNA was digested processively and synchronously with exonuclease III of E. coli (an enzyme splitting off only nucleotides from 3′ ends) and samples of double-stranded (ds) DNA, containing single-stranded (ss) ends of varying length (100–1800 nucleotides), were obtained. Digestion of nicked circular DNA (plasmid Col E 1) with the same enzyme yielded circular DNA containing ss regions inside the molecule. These DNA samples were investigated by means of derivative (differential) pulse polarography (DPP) in connection with mercury dropping electrode in a neutral medium. It was found that the presence of intramolecular ss regions in ds DNA was manifested by DPP peak III, i.e. in the same manner as the presence of free denatured DNA. The height of this peak increases linearly with the content of ss ends in ds linear DNA and DPP peak II (characteristic for ds DNA) does not increase in consequence of the formation of ss ends. This finding supports our notion about local changes in the conformation of DNA caused by various factors including temperature changes in the premelting region, and contradicts the assumption of other authors that polarographic reduction of DNA is independent of its structure and that a sufficient prerequisite for reduction is the adsorption of the DNA molecule at the electrode. ds DNA containing ss ends of varying length was investigated by means of linear sweep voltammetry in connection with stationary mercury electrode at neutral pH. It was found that the presence of ss ends influences markedly the course of the dependence of the voltammetric peak on the initial potential. An assumption has been formulated that this effect is connected with the fixation of the molecule ends in the electrode surface, which inhibits the surface denaturation of DNA.