Regulation of Catalysis by the Smallpox Virus Topoisomerase*□S

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoi-somerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoi-somerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp168 is a key regulator of the active site that maintains a