Covalent DNA binding by vaccinia topoisomerase results in unpairing of the thymine base 5' of the scissile bond.

We have used potassium permanganate to probe contacts between vaccinia DNA topoisomerase and thymine residues in its 5'-CCCTT downward arrow DNA target site. Two major conclusions emerge from the experiments presented: (i) permanganate oxidation of the +2T base of the scissile strand interferes with topoisomerase binding to DNA, and (ii) the +1T base of the scissile strand becomes unpaired upon formation of the covalent topoisomerase-DNA intermediate. Disruption of T:A base pairing is confined to the +1-position. Covalently bound DNAs that have experienced this structural distortion (such DNAs being marked by oxidation at +1T) are fully capable of being religated. We suggest that a protein-induced DNA conformational change is a component of the strand passage step of the topoisomerase reaction.