FIG. 6.

BPV DNA synthesis in membrane-depleted egg extracts. (A) Replication of double-stranded DNA in Xenopus HSS required E1 and a BPV origin of replication. E1 protein translated in Xenopus egg extracts was partially purified on a Q-Sepharose column, and increasing volumes of dialyzed E1 fraction, as indicated, were directly added to 15 μl of Xenopus HSS. Reaction mixtures were incubated for 3 h at 23°C after the addition of 150 ng of pSKori, containing the BPV minimal origin of replication (ori). The incorporation of [α-³²P]dCMP into an acid-insoluble form was measured by scintillation counting. (B) DpnI assay of reaction products obtained by incubating 150 ng of pSKori or pSVori DNA in 20-μl reaction mixtures containing 15 μl of HSS and 3.5 μl of E1 fraction for 3 h at 23°C. The reaction products were digested with DpnI and subjected to agarose gel electrophoresis. Both the ethidium bromide-stained gel and the autoradiogram are shown. F_I and F_II designate the migration positions of supercoiled monomer circle and nicked monomer circle, respectively.