Figure 5.

Topoisomerase activity of interspecies heterotetramers. (A) Supercoiling assay with the interspecies heterotetramers and the parent holoenzymes (25 nM). An amount of 300 ng relaxed pUC18 plasmid was used as substrate for the supercoiling assay. The topoisomers were resolved on a 1.2% agarose gel; (B) Decatenation activity of the parent and interspecies heterotetramers (25 nM) using 300 ng of kinetoplast DNA as substrate. The details of the reaction are given in ‘Materials and Methods’ section; (C) EMSA carried out with increasing concentrations (nM) of reconstituted hybrid heterotetramers (EAMB (EcGyrA + MsGyrB), MAEB (MsGyrA + EcGyrB) and MsGyr. 72-bp DNA was used as substrate. The DNA-bound complexes were resolved on a 5% native polyacrylamide gel. Arrows indicate the two complexes formed.