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 down arrow DNA target site. Two major conclusions emerge from the experiments presen ted: (i) permanganate oxidation of the +2T base of the scissile strand interferes with topoisomerase binding to DNA, and (ii) the +1T base o f the scissile strand becomes unpaired upon formation of the covalent topoisomerase-DNA intermediate. Disruption of T:A base pairing is conf ined to the +1-position. Covalently bound DNAs that have experienced t his structural distortion (such DNAs being marked by oxidation at +1T) are fully capable of being religated. We suggest that a protein-induc ed DNA conformational change is a component of the strand passage step of the topoisomerase reaction.