Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.
The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.
Virshup
D M
DM
Department of Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Kelly
T J
TJ
D000952:Antigens, Polyomavirus Transforming / Q000502:physiology; D002458:Cell Fractionation; D004265:DNA Helicases / Q000302:isolation & purification* / Q000502:physiology; D004261:DNA Replication; D006367:HeLa Cells; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D013539:Simian virus 40 / Q000235:genetics / Q000254:growth & development*; D014764:Viral Proteins / Q000302:isolation & purification* / Q000502:physiology; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D014764:Viral Proteins; D004265:DNA Helicases