Figure 2. Topoisomerase activity and CPT sensitivity of the double truncation LdTopIΔL/ΔS purified from yeast extracts.

A) Protein-dilution relaxation assay of LdTopIΔL/ΔS truncation (lanes 7 to 12) compared to wild type (lanes 1 to 6). 0.2 Micrograms of purified protein and a two-fold dilution series were incubated with 0.3 µg of RfI Φ X174 supercoiled DNA for 30 min at 37°C. B) Spot tests showing the sensibility to CPT of MBY3 yeast strain transformed with the “empty” pESC-URA vector (“mock”) or carrying the wild type or the double deletion LdTopIΔL/ΔS Exponentially growing cells in glucose, previously transformed with vectors carrying the corresponding topoisomerase encoding genes, were serially 10-fold diluted starting from OD₅₉₅ = 0.3. Five microliters were spotted on selective media under repressed (2% glucose) or induced (2% galactose) conditions, in the presence or absence of 15 µM and 30 µM CPT. “Mock” assays were performed under similar assay conditions using the pESC -URA vector without any insert C) Dose-response curves of these transformed yeast to different concentrations of CPT. A, B and C are pictures representatives of multiple experiments. D is the average of four independent experiments.