Abstract
The role that the carboxyl-terminal amino acids of Escherichia coli DNA topoisomerase I (Topo I) and III (Topo III) play in catalysis was examined by comparing the properties of Topo III with those of a truncated enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-terminal 605 amino acids of Topo III and the putative generalized DNA binding domain of Topo I. The deletion of the carboxyl-terminal 49 amino acids of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduces Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catalyzed 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. However, topoisomerase-catalyzed resolution of DNA replication intermediates was only stimulated 2-3-fold. Therefore, the carboxyl-terminal amino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the catalytic properties of these polypeptides.
Highlights
Escherichia coli contains two type I DNA topoisomerases, DNA topoisomerase I (1) and III (2, 3)
Overproduction and Purification of Topo 31Z, a Topoisomerase III-Topoisomerase I Chimera—In order to examine what was responsible for the different catalytic properties of topoisomerase I (Topo I) and Topo III, a chimeric molecule, Topo 31Z, was synthesized (Fig. 1A) that combined the first 605 amino acid residues of Topo III with the carboxyl-terminal amino acid residues of Topo I
The pattern of proteolysis is consistent between Topo 31Z and Topo I, suggesting that the proteolysis occurs within the identical carboxyl-terminal amino acid residues of the two enzymes
Summary
Escherichia coli contains two type I DNA topoisomerases, DNA topoisomerase I (1) and III (2, 3). 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.
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