Cell-free BKV DNA replication by recombinant BKV TAg. (A) DNA synthesis in the presence of recombinant BKV TAg was measured by incorporation of dNMPs into DNA. Increasing amounts (0, 7, 13, and 26 μg) of High Five cell extracts containing recombinant BKV TAg (about 0, 0.35, 0.65, and 1.3 μg, respectively) were added to HeLa hypotonic extracts in the presence of DNA with a BKV origin of replication (250 ng of B-B-B, ori+, bars 1 to 4) or empty vector (ori−, bars 5 to 8). (B) Cell-free DNA replication in the presence of BKV and SV40 TAg. DNA replication in the presence of 200 ng of purified recombinant BKV TAg or SV40 TAg as indicated was measured using 40 μg of HeLa extract and 500 ng of DNA containing the SV40 or BKV origin of replication (ori+) or an empty vector (ori−). Incorporation of dNMPs into DNA was measured by scintillation counting. The DNA synthesis was determined in duplicate and repeated three times. The averages from these experiments and the standard deviations are presented.