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. 2002 Jul;76(13):6518–6531. doi: 10.1128/JVI.76.13.6518-6531.2002

FIG. 5.

FIG. 5.

RPA catalyzes unwinding of the nicked minimal left-end origin. A DNA fragment containing the oriLTC 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.

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