Topoisomerase II can unlink replicating DNA by precatenane removal

We have analysed the role of topoisomerase II (topo II) in plasmid DNA repl ication in Xenopus egg extracts, using specific inhibitors and two-dimensio nal gel electrophoresis of replication products. Topo II is dispensable for nuclear assembly and complete replication of plasmid DNA but is required f or plasmid unlinking. Extensive unlinking can occur in the absence of mitos is. Replication intermediates generated in the absence of topo II activity have an increased positive super-helical stress (+Delta Lk), suggesting a d eficiency in pre-catenane removal. The geometry of replication intermediate s cut by poisoning topo II with etoposide and purified by virtue of their c ovalent attachment to topo II subunits demonstrates that topo II acts behin d the forks at all stages of elongation. These results provide direct evide nce for unlinking replicating DNA by precatenane removal and reveal a divis ion of labour between topo I and topo II in this eukaryotic system. We disc uss the role of chromatin structure in driving DNA unlinking during S phase .