Topology of the hRPA-dsMC complexes formed in the presence of magnesium probed by the <i>Ec</i> TopoI and the wheat germ Toposiomerase.

(A) Experimental scheme. The experiment is a succession of 6 steps: interaction between T-1 dsMC and hRPA in the presence of Mg2+ (1), electrophoresis under native conditions to separate nucleoprotein complexes from naked topoisomers (2), excision of Ca and Cb gel pieces after exposure of the gel under a 32P sensitive screen (3), “in gel” topoisomerase assay (4), DNA electro-elution (5) and electrophoresis under conditions that resolve topoisomers (6). (B) Determination of the topological state of the dsMC in the Ca and Cb complexes assembled on the T-1 topoisomer. Upper part of the figure: The species formed upon incubating hRPA (300 nM) with the T-1 topoisomer (0.5 nM) are resolved by electrophoresis on a 4% native polyacrylamide (29:1 = acrylamide:bisacrylamide mass ratio) gel made in TBMg (44.5 mM Tris-Base, 44.5 mM boric acid, 10 mM MgCl2). Lower part of the figure: The gel pieces corresponding to the Ca and Cb complexes are treated with Ec TopoI (Ec) or wheat germ Topoisomerase (wg) before performing an electro-elution to recover the DNA. The DNA recovered is precipitated and analyzed by electrophoresis on a chloroquine-containing gel (6% acrylamide; 29:1 = acrylamide:bisacrylamide mass ratio; 30 microg mL-1 of chloroquine; TBE 0.5x).