Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication.

Abstract

A 311 base pair DNA fragment, containing the simian virus 40 (SV40) origin of DNA replication, the early promoter and the tumor (T) antigen binding sites, was inserted into a bacterial plasmid and cloned. This recombinant plasmid, pSV01, binds to a purified T antigen in vitro and replicates in monkey cells when supplied with large T antigen. A series of deletion mutations was generated in the origin sequences of pSV01 DNA by mutagenesis in vitro. The replication of these mutant DNA in monkey cells was compared with their ability to bind to purified D2 protein. Mutant DNA, deficient in binding to D2 protein, exhibit reduced levels of replication in monkey cells. These findings provide biochemical evidence that the initiation of SV40 DNA synthesis may involve a direct interaction of T antigen with sequences at the origin of replication.