Abstract
Simian virus 40 (SV40) DNA replication has been reconstituted in vitro using purified proteins. SV40 large T antigen (TAg), DNA polymerase α-primase complex (pol α-primase), HeLa single-stranded DNA binding protein (HeLa SSB), and topo- isomerase I or II (topo I or II) produced extensive DNA synthesis when SV40 origin- containing DNA (ori+ DNA) was used as a template. Further addition of DNA ligase, RNase H, and a 5′→3′ exonuclease resulted in relaxed covalently closed circular DNA products (RFI’). Replication in vitro with purified proteins mimicked bona fide SV40 DNA replication in vivo by the following criteria: TAg dependency, functional SV40 origin dependency, aphidicolin sensitivity, leading and lagging strand semi-discontinuous synthesis, bidirectional replication from the origin, and covalently closed circular monomer products. Additionally, the species specificity of SV40 and polyoma virus DNA replication observed in vivo was also seen in vitro and can be attributed to the source of the pol α-primase.
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