FIGURE 4.

Molecular dissection of full-length PfTopoII to isolate a pure, stable functional PfTopoII construct with full ATP and Mg²⁺-dependent DNA decatenation activity. A, five new combinations of the three predicted PfTopoII domains were constructed (individually and in pairs) and tested for full topoisomerase functions. B, an autoradiogram demonstrating soluble expression of single-sized, radiolabeled truncated derivatives of PfTopoII. Solubility of the expressed proteins was confirmed by showing good representation in the soluble (S) fractions compared with the total (T) fraction in the wheat germ expression system. C, demonstration of decatenation activity in the PfTopoII-ΔCTD construct carrying NAD-CRD but lacking CTD. The decatenation function of PfTopoII-ΔCTD domain was stimulated by 50–100 mm NaCl, slightly less than that required for full-length PfTopoII (100–150 mm). D, demonstration of purity of PfTopoII-ΔCTD construct by SDS-PAGE, dependent DNA affinity chromatography (see “Experimental Procedures”).