title: The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.
abstract: A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.
authors.affiliation: Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
authors.forename: P R
authors.initials: PR
authors.lastname: Caron
authors.forename: P
authors.initials: P
authors.lastname: Watt
authors.forename: J C
authors.initials: JC
authors.lastname: Wang
mesh_terms: D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II