Figure 2.

oriL-dependent DNA replication products are of the θ-type. (A) The replication products that accumulate over the time using supercoiled pCB163 plasmid as the DNA template were analyzed in a native agarose gel stained with ethidium bromide. (B) Autoradiogram of the same gel. (C) The late replication intermediates formed are decatenated by the E. coli Topoisomerase III enzyme. Standard replication reactions with plasmid pCB163 as DNA template were performed for 15 min and then E. coli Topoisomerase III was added (varied in a 2-fold dilution series from 400 nM down to 50 nM), and reaction continued for 15 min. The deproteinized products were fractionated on a native agarose gel and autoradiographed. The abbreviations used are: C, control, pCB163 plasmid; LRI, late replication intermediates; FI, FII, FIII: Forms I, II and III of plasmid DNA, respectively.