P-Cell separation of replication proteins from Ad-infected cells. (A) Western blots of fractions I, IIA, IIB, and IIC with Pol δ, Pol ɛ, Pol α, Ad DBP, RFC, RPA, and PCNA antibodies. (B) Reconstitution of AAV DNA replication in vitro using fractionated Ad-infected-cell extracts. Standard replication reaction mixtures (30 μl) contained either crude Ad-infected-cell extract (C; 200 μg) or P-Cell fractions (I, 160 μg; IIA, 36 μg; IIC′, 30 μg; IID, 9 μg) and Rep78 (3.4 μg), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. The DNA products from each reaction were digested with DpnI and analyzed on 0.8% Tris-borate-EDTA (TBE)-agarose. md and dd indicate monomer duplex and dimer duplex DNA species, respectively, which are resistant to DpnI digestion. DNA products sensitive to DpnI digestion are denoted by the black line. (C) Optimization of AAV DNA replication with RFC and P-Cell fractions I and IIA. Standard replication reaction mixtures (15 μl) contained either crude Ad-infected-cell extract (C; 100 μg) or P-Cell fraction I (80 μg), RFC (0.5 μg), Rep78 (1.7 μg), and various amounts of fraction IIA (IIA total protein concentration, 6 mg/ml), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. Half of each reaction mixture was subjected to DpnI digestion and analyzed on 0.8% TBE-agarose; the remaining half was used to quantify [32 P]dAMP incorporation using a DE-81 filter binding assay (expressed as pmol of dAMP incorporated per 15 μl reaction mixture). The migration pattern and fragment size of radiolabeled HindIII-digested lambda DNA are indicated on the left.
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