THE ROLE OF THE CARBOXYL-TERMINAL AMINO-ACID-RESIDUES IN ESCHERICHIA-COLI DNA TOPOISOMERASE III-MEDIATED CATALYSIS

The role that the carboxyl-terminal amino acids of Escherichia coli DN A topoisomerase I (Topo I) and III (Topo III) play in catalysis was ex amined by comparing the properties of Topo III with those of a truncat ed enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-termin al 605 amino acids of Topo III and the putative generalized DNA bindin g domain of Topo I, The deletion of the carboxyl-terminal 49 amino aci ds of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduc es Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catal yzed resolution of DNA replication intermediates to a similar extent, Fusion of the carboxyl-terminal 312 amino acid residues of Topo I onto the truncated molecule stimulates topoisomerase-catalyzed relaxation 15-20-fold, to a level comparable with that of full-length Topo III. H owever, topoisomerase-catalyzed resolution of DNA replication intermed iates was only stimulated 2-3-fold. Therefore, the carboxyl-terminal a mino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the cat alytic properties of these polypeptides.