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

FIG. 6.

FIG. 6.

DNA replication with purified human proteins in the presence of BKV TAg and mouse cell extracts. (A) The replication of polyomavirus DNA was biochemically separated into three consecutive reaction steps: the unwinding, initiation, and elongation reactions. In the presence of RPA, topoisomerase I, ATP, and an ATP-regenerating system, viral TAg unwinds viral DNA at 37°C for 30 min (unwinding reaction). To synthesize primers at the unwound origin of DNA replication (initiation reaction), hPol α-primase and the three remaining ribonucleotides were added, and oligoribonucleotide primers are synthesized during the incubation at 37°C for 30 min, whereas no DNA can be synthesized since deoxynucleoside triphosphates are lacking. Finally, deoxynucleoside triphosphates, which include radioactively labeled dCTP to monitor DNA synthesis via scintillation counting, are added and DNA is synthesized at 37°C for 30 min (elongation reaction). Mouse cell extracts capable of supporting mPyV DNA replication were added to the reactions prior to the specified step (as indicated by the arrows). The addition of buffer served as control for the influence of salt and dilutions. (B) Results of the monopolymerase assay using BKV TAg and template containing the BKV origin of replication. (C) Results of the monopolymerase assay using SV40 TAg and template containing the SV40 origin of replication. In panels B and C, bars 1 and 2 represent dNMP incorporation into DNA in the absence and presence of TAg, respectively, but without mouse proteins. For bar 3, mouse cell extracts were added to reaction components before the addition of hPol α-primase and ribonucleotides (prior to unwinding of DNA). For bar 4, mouse cell extracts were added after the unwinding reaction but before the addition of hPol α-primase (prior to initiation of DNA replication). For bar 5, mouse cell extracts were added after initiation of DNA replication but before addition of deoxynucleoside triphosphates (prior to elongation). 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.