REQUIREMENTS FOR AND REGULATION OF ORIGIN OPENING OF PLASMID P1

Origin opening is essential for the initiation of DNA replication in t he theta mode and requires binding of initiator proteins. Using reacti vity to KMnO4 in vivo as an assay, we find that, like initiation, orig in opening of the Escherichia coli plasmid P1 requires the host initia tors DnaA and HU and the plasmid-encoded initiator RepA. The ability t o detect opening at the P1ori in vivo allowed us to study this activit y at various copy numbers in chimeric replicons, The opening was preve nted when the P1ori was cloned in high copy vectors or when excess Rep A binding sites (iterons) mere provided in trans. However, when RepA s upply was also increased, the opening was efficient. A further increas e in RepA prevented opening. Replication of an incoming pi under these conditions correlated with opening. These results demonstrate that in itiation is possible even at abnormally high origin concentrations and that oversupply of RepA, relative to iterons, can prevent replication by blocking origin opening. It appears that plasmid overreplication c an be prevented either by limiting RepA or by accumulating RepA at a r ate higher than that of the origin.