Interaction between hRPA and topoisomers of dsMCs in the presence of magnesium.

The dashed arrows indicate the direction of the electrophoretic migration. (A) The topoisomers of indicated topology are incubated with increasing amounts of hRPA. [hRPA] ranges from 0 to 1 microM (final concentration, 0; 30; 100; 300; 1000 nM). The reaction products are analyzed 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 hours. Three complexes form upon mixing a given topoisomer with hRPA: the hRPA-nicked dsMC complex, the Ca and the Cb complexes. (B) Same as (A) with a final concentration of hRPA of 300 nM. Samples were (“+” lanes) or not (“-”lanes) treated with proteinase K (Prot K) before their analysis by electrophoresis on a native polyacrylamide gel. The three protein-DNA complexes that assemble on the nicked, T-1, T-2, T-4 and T-4/-5 DNA are sensitive to Proteinase K.