Fig. 5

Mutations of ParE cavity residues affect DNA strand passage by topo IV. Complexes of topo IV reconstituted with ParE bearing cavity mutations at residues K291, R321, K346 or R353 exhibit greatly reduced DNA decatenation (a) and DNA relaxation (b) activities. a A representative decatenation experiment. For each panel, kinetoplast DNA was incubated at 37 °C for 1 h in decatenation buffer (40 mM Tris-HCl (pH 7.5), 6 mM MgCl₂, 10 mM DTT, 200 mM potassium glutamate, 50 µg ml⁻¹ BSA) with 1 mM ATP and S. pneumoniae topoisomerase IV reconstituted from a fixed amount of ParC (25 ng) (and in experiments not shown up to 2000 ng) and titrated with either wild-type or mutant ParE (as indicated) at 100, 20, 10, 5 and 2.5 ng (lanes 1–5, respectively in each panel). Reactions were terminated and the DNA products were separated and visualized by electrophoresis in 1% agarose gels. Lane C, no enzyme addition. kDNA, kinetoplast DNA; Mini denotes minicircle DNA released by topo IV. Intermediate bands are partially unlinked DNA species. b Relaxation assays for topo IV were carried as described for kDNA decatenation except supercoiled plasmid pBR322 DNA (400 ng) was used as substrate with the same level of ParC (25 ng) and wild-type or mutant ParE at 100, 40, 20, 10 and 5 ng (each panel lanes 1–5, respectively). DNA products were analysed on 1% agarose gels. Lane C, no enzyme addition. R and S, relaxed and supercoiled DNA, respectively