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. 2014 Jul 2;42(13):8578–8591. doi: 10.1093/nar/gku568

Figure 6.

Figure 6.

Relaxation and decatenation assays. (A) Relaxation assays of wild-type and double mutants of Microscilla marina (Mmar) topoisomerases VIII. Reactions were carried out without ATP and 200 ng of pBR322 was used as the substrate. Lane 1: control with no enzyme; lanes 2–7, wild-type (WT) with 1, 1.5, 2, 3, 6 and 12 pmol of enzyme, respectively; lanes 8–13, YY-FF double mutants with 0.5, 0.75, 1, 1.75, 3 and 6 pmol of enzyme, respectively. (B) Paenibacillus polymyxa (pPol) topoisomerase VIII relaxation assays with the wild-type enzyme; lanes 1–3, without ATP and with 10, 5, 2.5 pmol of enzyme, respectively, and lanes 4–6, with 1mM ATP and 10, 5, 2.5 pmol of enzymes, respectively; and with the D61A mutant lanes 7–9, without ATP, with 10, 5, and 2.5 pmol of enzyme, respectively, and lanes 10–12 with 1 mM ATP and 10, 5 and 2.5 pmol of enzyme, respectively. Supercoiled and relaxed (or open circular) topoisomers are noted as SC and R/OC, respectively. (C) Paenibacillus polymyxa topoisomerase VIII decatenation assays, with or without ATP; lane 1: no enzyme, lanes 2–4 with 0, 1 and 2 mM ATP, respectively and 20 pmol of enzyme. kDNA: kinetoplastid DNA.