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. 2009 Mar 18;83(11):5708–5717. doi: 10.1128/JVI.00300-09

FIG. 5.

FIG. 5.

DNA synthesis with purified human proteins in the presence of BKV and SV40 TAg. (A) Incorporation of dNMPs into DNA containing an SV40 (bar 2) or BKV (bar 4) origin of replication in the presence of 200 ng of the respective viral TAg and 100 ng of purified hPol α-primase, 50 ng topoisomerase I, and 1000 ng RPA was measured (monopolymerase DNA replication system). Vectors without a functional viral origin (ori−) served as negative controls (bars 1 and 3). (B) The effect of human and mouse proteins on DNA synthesis by hPol α-primase was determined with a BKV origin of replication. The incorporation of radioactive dNMPs using the BKV origin of replication as a template was measured in the presence of buffer but no additional proteins or with 15 μg human or mouse cell extracts (bars 1, 2, and 3, respectively). DNA synthesis in the presence of DNA lacking an origin of replication served as a negative control (bar 4). (C) The effect of human and mouse proteins on the DNA synthesis by human DNA Pol α-primase was determined with an SV40 origin of replication. The incorporation of radioactive dNMPs was determined in the presence of buffer but no additional proteins or with 15 μg human or mouse cell extracts (bars 1, 2, and 3, respectively). DNA synthesis in the presence of DNA lacking an origin of replication served as a negative control (bar 4). Incorporation of dNMPs into DNA was measured by scintillation counting. DNA synthesis was determined in duplicate and repeated three times. The averages from these experiments and the standard deviations are presented.