Replication of bovine papillomavirus type-1 origin-containing DNA in crude extracts and with purified proteins

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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 El 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 recon stituted 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.
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Issue Date
1994-06
Language
English
Article Type
Article
Keywords

SIMIAN VIRUS-40 DNA; CELL NUCLEAR ANTIGEN; SV40 T-ANTIGEN; INVITRO REPLICATION; RNA-POLYMERASE; BINDING-SITE; CLONED GENES; E2 PROTEINS; E1 PROTEIN; PHOSPHORYLATION

Citation

JOURNAL OF BIOLOGICAL CHEMISTRY, v.269, no.25, pp.17086 - 17094

ISSN
0021-9258
URI
http://hdl.handle.net/10203/65678
Appears in Collection
BS-Journal Papers(저널논문)
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