title: Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster. II. In vivo studies.
abstract: Genetic complementation, protein distribution, and in vivo enzymatic activity by carboxyl-terminal truncation mutations of the Drosophila enzyme were examined. Removal of more than 273 of the 1447 amino acids composing the full-length topoisomerase inactivates the enzyme in vivo and in vitro; removal of 227 amino acids or less has no apparent effect on the ability of the enzyme to substitute for a conditional lethal, or null mutation, of the Saccharomyces cerevisiae top2 gene. Four catalytically active mutants, from which 227 or 240 amino acids are deleted, define an intervening, critical region. Each mutant in this critical region displays different in vivo complementation activity ranging from complete complementation to noncomplementation. Deletion analysis revealed a potent nuclear localization signal within the most distal 60 amino acids, although this is apparently not the only functional signal sequence encoded in the enzyme. Subcellular fractionation and indirect immunofluorescence demonstrate that the truncated enzymes localize to the nucleus, albeit with reduced efficiency compared to wild type. The ability of these mutants, including a mutant in the critical region which does not complement, to catalyze the decatenation of replicated plasmids and the segregation of replicated chromosomes was also examined.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: D G
authors.initials: DG
authors.lastname: Crenshaw
authors.forename: T
authors.initials: T
authors.lastname: Hsieh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002875:Chromosomes; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013347:Subcellular Fractions
chemical_list: D004250:DNA Topoisomerases, Type II