Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. To address the mechanisms by which the viral DNA is stably propagated in the transformed cells, we have constructed a cell line CH04.15 expressing constitutively the viral proteins E1 and E2, that are required for initiation of viral DNA replication. We show that these viral proteins are necessary and sufficient for stable extrachromosomal replication. Using the cell line CH04.15, we have shown that the bovine papillomavirus‐1 (BPV‐1) minimal origin of replication (MO) is absolutely necessary, but is not sufficient for stable extrachromosomal replication of viral plasmids. By deletion and insertion analysis, we identified an additional element (minichromosome maintenance element, MME) in the upstream regulatory region of BPV‐1 which assures stable replication of the MO‐containing plasmids. This element is composed of multiple binding sites for the transcription activator E2. MME appears to function in the absence of replication but requires E1 and E2 proteins for activity. In contrast to, for example, Epstein‐Barr virus oriP, stably maintained BPV‐1 plasmids are not subject to once‐per‐cell cycle replication as determined by density labelling experiments. These results indicate that papillomavirus episomal replicators replicate independently of the chromosomal DNA of their hosts.

XylS, an AraC family transcription factor, positively regulates transcription of Pseudomonas putida TOL plasmid meta operon from the Pm promoter. A tandem of 15 bp homologous direct repeats, separated by 6 bp and overlapping with the -35 hexamer of the promoter, is required for the activation of Pm by XylS in vivo. In this study we have characterized specific binding of XylS to the Pm operator Om. XylS was overexpressed with an epitope tag in its N-terminus. Tagged XylS (N-XylS) was immunopurified and was shown to specifically bind to Om. We have used matrix-bound N-XylS in DNA footprinting and methylation interference experiments. Binding of N-XylS protects 44 bp in the Om region on both strands from DNase I digestion and generates hypersensitive sites (within the protected area) which lie on the same face of the DNA helix. Results of hydroxyl radical footprinting and methylation interference assays indicate that XylS binds along one side of the DNA and covers four helical turns. The protein has base-specific contacts in four adjacent major groove regions on the same helical face. Our data are in accord with the prediction of the presence of two separate DNA-binding units in an XylS molecule which are involved in base-specific contacts in two adjacent major-groove regions of a half-site. The direct repeat arrangement of the binding site and the mode of DNA binding of XylS are similar to the arrangement of recognition sites and the DNA contact pattern of AraC protein from Escherichia coli.