Figure 5. Agarose gel-based assay of DNA decatenation and relaxation by Methanosarcina mazei topo VI (MmT6).

On the left, a singly catenated (depicted by the linked green and orange circles), negatively supercoiled plasmid substrate is decatenated by 0.1–20 nM topo VI. The catenated, supercoiled plasmids vary in size and when decatenated, can be seen as two bands that migrate further (depicted by the separated green and orange circles) (Waraich et al., 2020). As they are also negatively supercoiled, the relaxation of the plasmids can be seen at topo VI concentrations ~10 fold higher (5 nM) than when full decatenation is seen (0.5 nM). This is further corroborated by a relaxation assay performed using negatively supercoiled pBR322* (right-hand gel), where relaxation activity is not detected until ~10 fold the MmT6 concentration (5 nM) necessary for decatenation. OC: open circular, Rel: relaxed and Sc: supercoiled. Both reactions were incubated for 30 min at 37 °C.