Drug sensitivity of top2::LEU2 yeast expressing the wild-type or mutated htopoIIα. Yeast growth is monitored in microtitre plates for 16 h in increasing concentrations of the indicated drug, as described in Materials and Methods. (A) Typical examples of growth curve data obtained for strain GA559 expressing htopoIIα E₁₅₅F in increasing concentrations of m-AMSA. (B–E) The OD₆₆₀ for a given strain which was recorded at a fixed time point (333 or 360 min as indicated) and is plotted as a function of increasing drug concentration [(B) m-AMSA; (C) o-AMSA; (D) VP16; (E) VM26] for the strains expressing the wild-type (diamond), K₁₂₃A (square) or E₁₅₅F (triangle) htopoIIα. The time point was chosen as being closest to half-maximal growth for the strain in the absence of drug, but resulting graphs were similar between 300 and 500 min growth [see panel (A)]. (F) Time to half-maximal yeast density is plotted as a function of VM26 concentration for strains expressing the wild-type, K₁₂₃A or E₁₅₅F form of htopoIIα. Error bars result from growth curves analysed in triplicate. Each experiment was performed three times.

Highly overexpressed htopoIIα N-terminal domains localise to the yeast nucleus. (A) After 16 h induction, cells expressing the indicated htopoIIα N-terminal domain or carrying an empty vector were formaldehyde fixed and immunostained for ytopoII (affinity purified αtopoII antibody is visualised in green) and for the htopoIIα domains (α-Myc, visualised in red). Co-localisation is shown in yellow. Insets show reactions in the absence of primary antibodies. Where indicated, the nuclear envelope is detected with mAb414 (α-pore, in red). (B) The level of overexpression of the htopoIIα N-terminal domains was determined in a strain expressing full-length wild-type htopoIIα from a single copy plasmid. Both constructs carry a single c-Myc epitope, which allows direct comparison of the relative levels of overexpression. Quantitation indicates at least 20-fold excess of the small domains over full-length protein.

Wild-type and two mutated htopoIIα complement the absence of yeast topoII. (A) The wild-type and point mutated forms of full-length htopoIIα cDNA were fused to c-Myc and 6-His epitope tags in a yeast expression vector (see Material and Methods). All constructs were sequenced to ensure that no additional mutations were introduced. (B) Radiolabelled [³H]VP16 and [α-³²P]ATP were used in a ligand binding assay performed in the presence of the indicated recombinant wild-type and mutated N-terminal domains (amino acids 1–440) of htopoII, as previously described (25). The ratio of binding efficiencies of both ligands was determined for each mutated form after each efficiency was normalised to the values obtained for the wild-type domain. (C) Individual transformants of strain GA559 expressing the indicated mutated form of htopoIIα were streaked on selective media in the presence (–ura –trp) or absence (–trp +FOA) of pBB6, a URA3-based vector expressing wild-type yeast TOP2. Controls include this same top2::LEU2 strain transformed with the empty vector pRS414 (–) or with vector pRS414 expressing wild-type htopoIIα (+). Growth is visible as a dark triangular patch. We see no dominant negative effects from the expression of any of the htopoIIα proteins in yeast. Only the wild-type and the point mutants E₁₅₅F and K₁₂₃A are able to substitute for yeast topoII to support mitotic growth in at least six of the eight colonies tested. (D) Full-length wild-type and the indicated mutant forms of htopoIIα are expressed at equal levels, both in the presence (left panel) and in the absence (right panel) of the endogenous yeast topoII. Western blots were performed on whole cell extracts of the indicated transformants using mouse anti-Myc (9E10) and rat anti-p42^{RNase H}, an abundant cytosolic protein that serves as an internal standard. The negative control strain (– control) carries an empty vector, while the positive control (+) expresses both yeast and human topoII enzymes.

Overexpression of the htopoIIα N-terminal domain confers drug resistance in yeast. (A) Scheme of the fusion between htopoIIα wild-type or mutant N-terminal domains and the NLS. (B) Time required to reach half-maximal culture density (t_{½ max}) was determined for the transformed yeast strain GA1072, expressing the indicated htopoIIα N-terminal domains. The contents of eight microtitre wells of each transformant were analysed by western blotting with antibodies against Myc or p42^{RNase H}. (C) The increase in t_{½ max} is shown as a function of VP16 concentration for GA1072 transformants expressing the indicated htopoIIα N-terminal domain: no protein (diamond), N-term.NLS fusions wild-type (square), E₁₅₅F (triangle), K₁₂₃A (cross) and G₁₆₄V (disc). Note that GA1072 carries transporter mutations and is more sensitive to VP16 than normal yeast strains. The t_{½ max} values in the absence of drug were normalised to the strain carrying an empty vector. Error bars result from growth curves analysed in triplicate. Each experiment was performed three times. (D) The levels of expression of both the human and the yeast topoII were analysed in yeast. The strains GA1659 (expressing only wild-type htopoIIα, left panel) and GA1072 (expressing endogenous ytopoII, right panel) were grown and counted when harvested. Whole cell extracts were prepared and analysed by quantitative western blotting with anti-pentaHis or anti-ytopoII antibodies, respectively. Increasing amounts of the yeast transcription regulator Sir2-6His and yeast topoII, both purified to homogeneity, were used as standards for the quantification. * indicates a non-specific signal obtained with the antibody raised against ytopoII.

A model for a regulatory role of the N-terminal domain in etoposide action. (A) To explain the drop in VP16 sensitivity conferred by overexpression of the wild-type htopoIIα N-terminal domain (dark circle) in yeast, we propose that it sequesters the drug (black squiggle) and thereby enhances resistance of the transformed strain. This is compromised by mutations that interfere with VP16 binding. (B) Under drug-limiting conditions that may occur in yeast, the N-terminal and the core domain appear to compete intra- or intermolecularly for etoposide (see Fig. 2). We propose that the N-terminal site functions mainly when topoII is not DNA (double helix) bound. Drug association with this domain appears not to induce a cytotoxic cleavage. (C) When mutations that reduce etoposide binding are introduced into the N-terminal domain, more inhibitor may become available to the site in the core domain, increasing cytotoxicity.