TOPOISOMERASE-IV CAN SUPPORT ORIC DNA-REPLICATION IN-VITRO

Escherichia coil has two type II topoisomerases, DNA gyrase and topois omerase IV (Topo IV). Topo IV is required for the decatenation of the linked daughter chromosomes at the terminal stages of DNA replication, whereas gyrase, because of its ability to convert to negative superco ils the positive supercoils generated by replication fork progression in a circular chromosome, is required to support nascent chain elongat ion. Using an oriC DNA replication system in vitro, we show that Topo IV, which can relax positive supercoils, can also support replication fork progression. This activity is only observed at substoichiometric ratios of Topo IV to template, at higher ratios, the template becomes relaxed and initiation of DNA replication cannot occur. Topo IV was ca pable of supporting bidirectional DNA replication from oriC, although, unlike the case with gyrase, some templates apparently replicated uni directionally. This suggests that either gyrase itself or a certain mi nimum superhelical density is required for proper initiation of DNA re plication from oriC.