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