Interference of the simian virus 40 origin of replication by the cytomegalovirus immediate early gene enhancer: Evidence for competition of active regulatory chromatin conformation in a single domain

Replication origins are often found closely associated with transcription r egulatory elements in both prokaryotic and eukaryotic cells. To examine the relationship between these two elements, we studied the effect of a strong promoter-enhancer on simian virus 40 (SV40) DNA replication. The human cyt omegalovirus (CMV) immediate early gene enhancer-promoter was found to exer t a strong inhibitory effect on SV40 origin-based plasmid replication in Co s-1 cells in a position- and dose-dependent manner. Deletion analysis indic ated that the effect was exerted by sequences located in the enhancer porti on of the CMV sequence, thus excluding the mechanism of origin occlusion by transcription. Insertion of extra copies of the SV40 origin only partially alleviated the inhibition. Analysis of nuclease-sensitive cleavage sites o f chromatin containing the transfected plasmids indicate that the chromatin was cleaved at one of the regulatory sites in the plasmids containing more than one regulatory site, suggesting that only one nuclease-hypersensitive site existed per chromatin. A positive correlation was found between the d egree of inhibition of DNA replication and the decrease of pi cleavage freq uency at the SV40 origin. The CMV enhancer was also found to exhibit an inh ibitory effect on the CMV enhancer-promoter driving chloramphenicol acetylt ransferase expression in a dose-dependent manner. Together these results su ggest that inhibition of SV40 origin-based DNA replication by the CMV enhan cer is due to intramolecular competition for the formation of active chroma tin structure.