MUTATIONS WITHIN A CONSERVED REGION OF VACCINIA TOPOISOMERASE AFFECT THE DNA CLEAVAGE-RELIGATION EQUILIBRIUM

The segment of the vaccinia DNA topoisomerase from residues 143 to 167 (VGLLTLKNKHIEISPDEIVIKFVGK) is conserved in other members of the euka ryotic type I topoisomerase family. In order to gauge the function of this region, we performed a mutational analysis in which 23 of 25 posi tions were substituted by alanine. Several non-alanine mutations were also studied. Purified wild-type and mutant proteins were compared wit h respect to their activities in relaxing supercoiled DNA and in singl e-turnover strand cleavage. Lys167, an invariant residue, was judged e ssential for catalysis, insofar as alanine replacement resulted in a 1 00-fold decrement in specific activity. Alanine substitutions for inva riant residues Gly144 and Gly166 were well-tolerated, but a G144R muta tion inactivated the enzyme and G166R reduced activity by two orders o f magnitude. More modest effects of other mutations were demonstrated by kinetic analysis of the single-turnover DNA cleavage and religation reactions and by studies of covalent adduct formation under equilibri um conditions. Mutations G144A and T147A elicited a shift in the cleav age-religation equilibrium toward the non-covalently bound state; this was caused by slowing of the forward cleavage reaction. Mutations F16 4A, G166A, G166R, K167A, and K167R produced opposite effects on reacti on equilibrium, resulting in higher levels of covalent complex formati on. We suggest that invariant residues F164, G166, and K167, constitut e part of the active site of the enzyme. (C) 1996 Academic Press Limit ed