Replication of bovine papillomavirus type 1 origin-containing DNA in crude extracts and with purified proteins.
The in vitro replication of DNA containing the bovine papillomavirus (BPV-1) origin has been carried out with cell-free extracts from mouse FM3A and human HeLa cells. DNA synthesis required the E1 protein, the minimal origin of replication (nucleotides 7911-22 of the BPV-1 genome), and, at low levels of FM3A extract, the addition of the human single-stranded DNA-binding protein (also called RP-A or RF-A). The E2 protein was not absolutely required, but could stimulate DNA synthesis at low levels of E1. DNA synthesis was also reconstituted using purified proteins from HeLa cells. These protein factors included human single-stranded DNA-binding protein, topoisomerase I, and DNA polymerase (pol) alpha-primase complex. At low concentrations of pol alpha-primase complex, the formation of high molecular weight products was dependent on the addition of DNA polymerase delta holoenzyme containing proliferating cell nuclear antigen and activator 1, also called RF-C. We have overexpressed and isolated the E1 protein from bacteria. This protein also supported BPV DNA synthesis, both in crude extracts and with purified proteins suggesting that E1 phosphorylation is not required for BPV DNA replication in vitro.
Müller
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Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, New York, New York 10021.
Seo
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Hurwitz
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D000818:Animals; D001483:Base Sequence; D052688:Bovine papillomavirus 1 / Q000235:genetics*; D002474:Cell-Free System; D019915:DNA Primase; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D017931:DNA Primers; D004268:DNA-Binding Proteins; C069814:E1 protein, Bovine papillomavirus; C051550:E2 protein, Bovine papillomavirus; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase