title: The role of the carboxyl-terminal amino acid residues in Escherichia coli DNA topoisomerase III-mediated catalysis.
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.
authors.affiliation: Molecular and Cell Biology Program, University of Maryland, Baltimore, Maryland 21201, USA.
authors.forename: H L
authors.initials: HL
authors.lastname: Zhang
authors.forename: S
authors.initials: S
authors.lastname: Malpure
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: H
authors.initials: H
authors.lastname: Hiasa
authors.forename: R J
authors.initials: RJ
authors.lastname: DiGate
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004264:DNA Topoisomerases, Type I