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 temperaturedependent 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. ). In vitro, the DNA transport reaction catalyzed by the yeast enzyme is manifested in interconversions between topological isomers of DNA rings: relaxation of positively and negatively supercoiled DNA, catenation and decatenation, and knot formation and removal. In vivo, the enzyme and its homologs have been shown to unlink multiply intertwined pairs of newly replicated DNA rings (18) and to remove positive and negative supercoils generated by transcription (24). Whereas the supercoiling-relaxation function of the enzyme can be carried out by DNA topoisomerase I, the unlinking of multiply intertwined DNA rings or chromosomal loops without nicks or gaps is a unique function of the type II enzyme; thus, inactivation of the type II enzyme invariably leads to cell inviability (25, 59), which is presumably a consequence of the failure of intertwined pairs of chromosomes to segregate properly during mitosis (18,29,60).In Schizosaccharomyces pombe cells lacking DNA topoisomerase II, chromosome condensation is also blocked (58). The involvement of the enzyme in chromosome condensation is supported by in vitro experiments with cell extracts (3,43) and by results on the time dependence of the cellular localization of the enzyme in Drosophila embryos undergoing synchronized cell division (7, 56). The precise role of the enzyme in chromosomal condensation, namely, whether the enzyme enables or facilitates the process through its catalysis of DNA breakage, passage, and rejoining or participates as a stoichiometric component of the nucleoprotein product, is not known. Th...

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

“…The constructs used are C-terminal truncations, of which all but the shortest leave the active site of the enzyme intact and retain the conserved regions homologous to type II topoisomerases, including GyrA and GyrB of E. coli (see Figure 2). The truncations remove the major sites of casein kinase II modification in the C-terminal 200 aa of the enzyme (indicated as arrows above the gene), and in all but the truncation at aa 1236, nuclear localisation signals (between aa 1166 and 1208, Caron et al, 1994) are removed. All constructs are inserted in the same multicopy vector with a galactose-inducible promoter, which allows differential levels of transcription depending on the carbon source, i.e.…”

“…In yeast two truncated forms, terminating at aa 1235 and aa 1191, respectively, were shown to confer a low level of drug resistance to amsacrine (at 10 ,g ml-'; Wasserman and Wang, 1994a). In contrast to our conditions, these mutant forms were selected for their ability to support growth as well as confer drug resistance, hence they were at least partially nuclear and enzymatically active (Wasserman and Wang, 1994a;Caron et al, 1994). Our screen for dominant drug resistance in yeast can be readily applied to domains of the human topoisomerase Ilx gene, since we are not dependent on complementation of the top2's deficiency.…”

“…Lee et al, 1992;Liu et al, 1991), which may explain the resistance phenotype, since less DNA damage might be provoked by less active enzymes. The third group is more enigmatic, since it was shown that the Cterminal 220 aa of DNA topoisomerase II is not essential for the enzymatic activity of the enzyme in Schizosaccharomyces pombe (Shiozaki and Yanagida, 1991), Drosophila melanogaster or Saccharomyces cerevisiae (Caron et al, 1994;Crenshaw and Hsieh, 1993a,b), although it contains major regulatory phosphoacceptor sites. Indeed, phosphorylation has been demonstrated to modulate the enzymatic activity of topoisomerase II in several species (reviewed in Cardenas and Gasser, 1993).…”

Structure of the Topoisomerase IV C-terminal Domain

“…Multiple sequence alignment revealed that, although most functional domains are conserved in all type IIA enzymes, the highly charged C-terminal domain (CTD) of the eukaryotic Topo has no detectable sequence similarity with the corresponding regions in either DNA gyrase or Topo IV (20). Instead of involving catalysis, various studies have suggested that the intracellular localization of eukaryotic Topo IIA is regulated by its CTD (21)(22)(23)(24). In contrast, the two prokaryotic enzymes are assembled from two closely related subunits.…”

Hypophosphorylation of Topoisomerase IIα in Etoposide (VP‐16)‐resistant Human Carcinoma Cell Lines Associated with Carboxy‐terminal Truncation

“…10) In contrast, C-terminal truncation of the S. pombe topoisomerase II to amino acid 1198, which removes most of the potential CKII phosphoacceptor sites, partially complements an S. pombe top2ts mutation and a null mutant, allowing conditional growth, yet fails to complement another top2ts mutation. 11) Surprisingly, protease digestion studies show that a proteolytic core of the enzyme that extends only to amino acid 1220 retains the full decatenation activity of topoisomerase II. Similarly, a truncation of the S. cerevisiae enzyme to amino acid 1167, produces a topoisomerase II dimer that is fully active in vitro, but which complements a top2 disruption poorly in vivo.…”