MUTATIONAL ANALYSIS OF VACCINIA DNA TOPOISOMERASE DEFINES AMINO-ACID-RESIDUES ESSENTIAL FOR COVALENT CATALYSIS

The eukaryotic family of type I DNA topoisomerases includes the nuclea r type I enzymes and the enzymes encoded by vaccinia and other poxviru ses. The small size of the vaccinia topoisomerase (314 amino acids as compared to 765-972 amino acids for the cellular enzymes) makes it lik ely that this protein constitutes the minimal functional unit of a euk aryotic type I enzyme and provides an opportunity for a comprehensive structure-function analysis through mutagenesis. Two protein subregion s were targeted for mutagenesis in the present study. The role of the Ser-Lys-X-X-Tyr sequence present at the active site of all family memb ers was examined by replacing each conserved residue with alanine. Ala nine substitution at the active site Tyr abrogated topoisomerase activ ity. In contrast, mutations at Ser-270 and Lys-271 had no effect on en zyme activity. The region of the vaccinia topoisomerase from amino aci ds 126-142 (MFFIRFGKMKYLKENET) is highly conserved and contains a resi due, Gly-132, shown previously to be essential. Twenty-nine different mutations were generated in this region, with at least one substitutio n at each position. Point mutations were identified at three positions , Arg-130, Tyr-136, and Leu-137, which either abrogated or severely re duced DNA relaxation. The effects on activity could be attributed to a defect in formation of the covalent intermediate. Alterations of 13 o ther amino acids, including conserved residues, had little or no effec t on topoisomerase activity.