FIG. 5.

RPA catalyzes unwinding of the nicked minimal left-end origin. A DNA fragment containing the oriL_TC sequence, ³²P labeled at its 3′ ends, was incubated with 3 mM ATP in the presence or absence of NS1 (100 ng) and PIF (25 ng) and increasing amounts of either recombinant RPA (0.125, 0.25, 0.5, and 1 μg) or E. coli SSB (0.125, 0.25, 0.5, and 1 μg), as indicated at the top of the gel. For lane NS1+PIF (boiled), the nicking reaction mixture was boiled immediately before electrophoresis in an SDS-polyacrylamide gel. Note that only covalent NS1-DNA complexes are significantly retarded in this assay. A schematic model of the nicking-coupled unwinding reaction is depicted at the right of the gel autoradiograph. The asterisks indicate the position of the ³²P label on each strand of the origin DNA.