Analysis of nonpermissivity in mouse cells overexpressing simian virus 40 T antigen.

To analyze the nature of the nonpermissivity of mouse cells for simian virus 40 (SV40) DNA replication, we isolated mouse cells producing SV40 T antigen (Tag) at levels equal to or greater than that found in COS1 cells. These mouse cells were nonpermissive for the replication of exogenously added SV40 DNA, although purified Tag isolated from these cells was able to support SV40 DNA replication in vitro. Furthermore, when mouse cells expressing Tag were fused to monkey cells, SV40 DNA replication was observed.

T antigen and template requirements for SV40 DNA replication in vitro.

A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis.

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA.

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. II. Purification and characterization of a host protein required for the replication of DNA templates devoid of the terminal protein.

A host protein, which is required for the replication of a plasmid DNA (pLA1), has been purified from extracts of uninfected HeLa nuclei. This plasmid DNA contains the origin of adenovirus DNA replication but lacks the 55,000-dalton terminal proteins. The purified host protein has been designated factor pL.

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences.

An in vitro system which replicates plasmid DNA containing the replication origin of adenovirus DNA has been established. Replication of plasmid pLA1 DNA, which contains the left-hand terminus (0-9.4 map units) of adenovirus serotype 5 DNA but which lacks the 55,000-dalton terminal protein, is initiated by a protein-primed mechanism in a manner similar to that found with adenovirus DNA.