Cleavage of Supercoiled DNA by Deoxyribonuclease I in Solution and at the Electrode Surface

Abstract

Cleavage of supercoiled DNA by deoxyribonuclease I (DNase I) in solution and at the surface of the mercury electrode was studied by means of AC voltammetry. This technique produces peak 3 which is produced only by DNAs containing free ends (such as linear double-stranded and single-stranded DNAs and open circular DNAs) but not by covalently closed circular (ccc) DNAs. Formation of a single interruption of the sugar-phosphate backbone in the ccc supercoiled (sc) DNA results in formation of peak 3. Peak 1 is produced by both ccc DNA molecules as well as by DNAs containing free ends; changes in height of this peak occur due to DNA cleavage. We show that the kinetics of the cleavage of DNA in solution and at the electrode surface substantially differ suggesting restricted accessibility of the surface-confined DNA for the interaction with the enzyme. Cleavage of the immobilized DNA is remarkably influenced by the potential of the electrode surface. At positively charged surface the enzymatic reaction is inhibited in its initial stage while moderately negative charges stimulate the cleavage of the immobilized DNA by DNase I.