title: Linker insertion mutagenesis of Drosophila topoisomerase II. Probing the structure of eukaryotic topoisomerase II.
abstract: The sequences of all type II DNA topoisomerases, and possibly some of their key structural features, are conserved. The N-terminal and middle regions of the eukaryotic DNA topoisomerase II are homologous to the bacterial gyrase subunits B and A, respectively, and the hydrophilic C-terminal region is more divergent among these enzymes. To gain further insights into the structure of eukaryotic topoisomerase II, we constructed 23 linker insertion mutants of Drosophila DNA topoisomerase II. These mutant proteins were expressed in a heterologous yeast system, in which we have previously demonstrated that Drosophila DNA topoisomerase II could be functionally expressed and complement yeast top2 mutations. The linker insertion mutants were characterized genetically by testing for complementation of yeast top2ts mutation at the non-permissive temperature and complementation of yeast top2 null mutation using a color sector assay. We also partially purified the mutant proteins and examined their enzymatic activity by unknotting the P4 knotted DNA. There appears to be a good correlation between the in vivo and in vitro activities. There are nine fully active, six partially active, and eight negative linker insertion mutants. All five linker insertion mutants in the C-terminal region are active and two linker insertion mutants located in the junction of the two regions homologous to gyrB/gyrA subunits are also active. In addition, we also mapped the trypsin-sensitive sites in Drosophila DNA topoisomerase II. The C-terminal region is extremely sensitive to trypsin digestion. Another major trypsin-sensitive site is located between Lys406 and Thr407, which is near the protease sites also observed in the bacterial gyrB subunit and yeast topoisomerase II. We discuss the possible structural and functional implications of these results.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
authors.forename: M P
authors.initials: MP
authors.lastname: Lee
authors.forename: T S
authors.initials: TS
authors.lastname: Hsieh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004330:Drosophila / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014357:Trypsin
chemical_list: D014357:Trypsin; D004250:DNA Topoisomerases, Type II