Topology of the DNA in the hRPA-dsMC complexes formed in the presence of magnesium.

(A) Experimental scheme. The experiment is a succession of 5 steps: interaction between dsMC and hRPA in the presence of Mg2+ (1), electrophoresis under native conditions to separate nucleoprotein complexes from naked dsMCs (2), excision of Ca and Cb gel pieces after exposure of the gel under a 32P sensitive screen (3), electro-elution (4) and electrophoresis under conditions that resolve topoisomers (5). (B) Topology of the DNA in the Ca and Cb complexes. Upper part of the figure: hRPA (final concentration, 300 nM) is incubated with the T-2 (left panel), T-5 (middle panel) or T-6 (right panel) topoisomer (final concentration, 0.5 nM) and the different molecular species are resolved by electrophoresis on a native 4% polyacrylamide (29:1 = acrylamide:bisacrylamide mass ratio) gel made in TBMg (44.5 mM Tris-Base, 44.5 mM boric acid, 10 mM MgCl2). Electrophoresis is performed at 150 V, 4°C and for 4 h. Lower part of the figure: The DNAs contained in the gel bands corresponding to the Ca and Cb complexes are electro-eluted 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). The dsMC interacting with hRPA is either T-2 (left part of the figure), T-5 (middle part of the figure) or T-6 (right part of the figure).