Study of plasmid replication in Escherichia coli with a combination of 2D gel electrophoresis and electron microscopy

We studied theta-mode DNA replication in p15A-based Escherichia coli plasmids by analyzing their replication intermediates using a combination of neutral agarose 2D gel electrophoresis and electron microscopy. Our analysis: (1) confirms the original assignment of various features of the 2D gel pattern; (2) shows that while one replication fork progresses around the plasmid DNA, the other is immobile, as if the replication were unidirectional; and (3) reveals that termination often occurs at a location away from the replication origin, suggesting that the replication of our plasmids is, in fact, bidirectional, the two forks being active at different times. (C) 1997 Academic Press Limited.