Supplementary Figure 3: Evidence that supercoiling occurs via a translocation-dependent mechanism.

(a, b) Schematic representation of mechanistic models for PICH-mediated positive supercoiling. PICH is depicted as a red circle. Positive and negative torsional stress induced by PICH translocation are indicated by red “+” and “-” symbols (Translocation models; b). The (-) and (+) symbols indicate the topology of DNA segments resulting from PICH wrapping DNA (Wrapping model; a) or from PICH translocation (Translocation model; b). Net supercoiling level (ΔLk) of each intermediate is indicated in the gray bar below each panel. (a) In the wrapping model, the bound DNA segment might be wrapped around PICH in a positive supercoiling conformation, leading to the introduction of compensatory negative supercoils in the unbound DNA segment. Type IA (upper panel) and Type IB or II topoisomerases (lower panel) then relax the negative supercoils, resulting into the introduction of net positive supercoiling. (b) In the translocation model, and according to the twin-supercoiling domain model, PICH translocation might lead to the redistribution of DNA torsional stress leading to the accumulation of unconstrained positive and negative supercoiling, respectively, in front and behind PICH. Type IA (upper panel) would only relax the negative supercoils, resulting in the introduction of net positive supercoiling. Type IB or II topoisomerases (lower panel) would relax both positive and negative supercoils, leading to no net alteration in supercoiling. A topological barrier induced by PICH is depicted as a black line and red square. (c-f) Representative 1D agarose gel electrophoresis showing the influence of PICH on the activity catalyzed by increasing concentrations of wgTop1 (c), ecTop1 (d), ecTop3 (e), and TRR (f) using a negatively supercoiled plasmid as a substrate. Agarose gel electrophoresis was performed in the absence (neutral) (c, d) or in the presence of chloroquine (e, f) in order to reveal variations of topology within the negatively supercoiled topoisomers. For each experiment, open circular (oc), linear (lin), relaxed (rel), negatively (-) and positively (+) supercoiled plasmids were used as markers. For each panel, representative images out of at least 3 independent experiments are presented. Each independent experiment lead to similar results. (g) Additional examples of representative AFM topographs of open circular DNA plasmids incubated in the presence of PICH and ATPγS. Height scale bar is common for all images and is shown on the right. XY scale bar is 100 nm. Images representative of at least 10 images collected for 3 independent experiments are presented. All experiments lead to similar results.