THE C-TERMINAL DOMAIN OF SACCHAROMYCES-CEREVISIAE DNA TOPOISOMERASE-II

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of t he 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 polypep tides; truncation of the C terminus to Ile-1220 has little effect on t he function of the enzyme in vitro or in vivo, whereas truncations ext ending beyond Gln-1138 yield completely inactive proteins. Several mut ant 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 t o contribute to the physiological dysfunction of these proteins; the a bility of these mutant proteins to relax supercoiled DNA in vivo shows , however, that at least some of the mutant proteins are present in th e nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intrace llular DNA, all mutants that do not complement the temperature-depende nt lethality and high frequency of chromosomal nondisjunction of top2- 4 were found to lack decatenation activity in vivo. The plausible role s of the DNA topoisomerase II C-terminal domain, in addition to provid ing a signal for nuclear localization, are discussed in the light of t hese results.