Abstract
An in vitro replication system has been used to study the origin and direction of replication of the covalently closed, circular DNA of plasmid RSF1030, a nonconjugative R factor. We have enriched for replicative intermediates in these studies either by isolating them on the basis of their unique structure or by limiting the extent of synthesis in the in vitro system. Circular molecules that have replicated to various extents migrate to characteristic positions in agarose gels, thus providing a rapid and efficient method for isolating partially replicated forms. Alternatively, replicative intermediates can be isolated directly from reaction mixtures that contain dideoxyTTP (ddTTP), a compound that limits the average extent of synthesis in vitro. Electron microscopic analysis of such intermediates linearized with either Hpa I or BamHI indicates that RSF1030 replicates in vitro from a unique origin located 70% from one end of Hpa I-cleaved molecules and 47% from the BamHI site. The unidirectional mode of replication has been confirmed by the order in which the six HincII fragments of RSF1030 DNA are labeled in vitro when synthesis is limited to various extents with ddTTP. Finally, a physical map of RSF1030 has been constructed using the restriction endonucleases BamHI, Hpa I, and HincII, and the origin and direction of replication have been defined relative to the map.