MECHANISM OF DNA TRANSESTERIFICATION BY VACCINIA TOPOISOMERASE - CATALYTIC CONTRIBUTIONS OF ESSENTIAL RESIDUES ARG-130, GLY-132, TYR-136 AND LYS-167

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxat ion of supercoiled DNA by cleaving and rejoining DNA strands through a (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167, Arg-130, Gly-132 and Lys-167 are conser ved in all members of the type IB topoisomerase family. Tyr-136 is con served in all poxvirus topoisomerases, We show that Arg-130 and Lys-16 7 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the a lanine mutant, We propose that both basic residues interact directly w ith the scissile phosphate at the topoisomerase active site, Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects o n religation rate, Gly-132 and Tyr-136 are suggested to facilitate a p re-cleavage activation step, The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural si milarities to site-specific recombinases.