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. 2022 Jan 25;11:e67021. doi: 10.7554/eLife.67021

Figure 3. ATPase activity and DNA binding of topo VI are stimulated by supercoiled DNA.

(A) The ATPase activity of topo VI over time, measured using radioactive ATP. Assays were performed at 21 °C, using 1 μM topo VI, 430 nM pBR322* that was negatively-supercoiled (blue), linear (green), or relaxed (orange), and 450 μM [γ-32]-ATP. (B) Agarose-gel based relaxation time course performed under the same conditions as A, using the same topo VI:DNA ratio (1:2.3), but with non-radioactive ATP. In A, the ATPase rate on supercoiled DNA plateaus around 15 min, which corresponds to the DNA being fully relaxed by topo VI, as shown in B. Samples were run on a 1% (w/v) native agarose gel for 15 hr at ~2 Vcm–1, stained with 0.5 μg/mL ethidium bromide and imaged under UV illumination. (C) The ATP-independent DNA binding activity of topo VI, measured using a nitrocellulose membrane capture technique, with either negatively-supercoiled (-sc), positively-supercoiled (+ sc), relaxed (rel) or linearised (lin) pBR322* (± SEM). DNA concentrations measured using A260. (D) The ATP-independent DNA binding activity of topo VI, on either positively- (red) or negatively-supercoiled (blue) topoisomers of pBR322* measured using a nitrocellulose membrane capture technique. Bound and unbound DNA samples were run on a 1% (w/v) native agarose gel for 15 hr at ~2 Vcm–1, stained with 0.5 μg/mL ethidium bromide and imaged under UV illumination. The intensity of the bands were measured using ImageJ and the relative dissociation constants (Kd± SEM) for each topoisomer calculated as described in Litwin et al., 2015. (E) DNA cleavage activity of topo VI using negatively- and positively-supercoiled pBR322*. Topo VI concentration was varied from 5 to 80 nM and incubated with 4 nM pBR322*, 1 mM ADPNP, 10 mM MgCl2, at 37 °C for 30 min. All samples were treated with 1 mg/mL proteinase K and 0.2% SDS, then run on a 1% (w/v) native agarose gel for 10 hr at ~2 Vcm–1, stained with 0.5 μg/mL ethidium bromide and imaged under UV illumination.

Figure 3—source data 1. Source data is in the file Figure 3.

Figure 3.

Figure 3—figure supplement 1. ATPase activity of M. mazei topo VI with different DNA species.

Figure 3—figure supplement 1.

ATPase rates were measured using a PK/LDH linked assay (Feng et al., 2021) with 1.2 µM topo VI in the presence of 177 nM DNA at 37 °C.
Figure 3—figure supplement 1—source data 1. Source data is in the file Figure 3—figure supplement 1.