Negative Control of DNA Replication Revealed in Composite Simian Virus 40-Bovine Papillomavirus Plasmids

Abstract

We have begun an analysis of DNA sequences that function in the control of DNA replication in eukaryotic cells. Bovine papillomavirus (BPV) replicates as an extrachromosomal nuclear plasmid, duplicating each viral genome in synchrony with the host-cell DNA once and only once per cell division. Simian virus 40 (SV40) replication, however, is unresponsive to cellular controls, so the viral DNA replicates exponentially within the host-cell nucleus. To approach our study of the mechanisms of replication control, we designed a model system consisting of SV40 and BPV DNA sequences linked to create a hybrid replicon. We have obtained evidence that a main feature of copy-number regulation in the composite SV40-BPV plasmid is the imposition of dominant negative control encoded by BPV, which overrides the runaway replication induced by the positive factor, SV40 large tumor (large-T) antigen. Using a transient replication assay, we have previously been able to define the elements in the BPV genome that are sufficient to establish controlled replication in composite SV40-BPV plasmids (Roberts and Weintraub, 1986). These elements consist of two cis-acting sequences that are closely linked to BPV replication origins and a third element that acts in trans. The latter is encoded within the 5′ part of the E1 open reading frame (ORF) of the BPV genome and is physically and functionally separable from the positive replication factor encoded within the 3′ part of the same ORF. The controlled replication of SV40-BPV composite plasmids has enabled us to create permanent COS cell lines that stably maintain these plasmids as episomes.