Topo IV is required to allow replisomes to converge and complete replication on the chromosome.

The ability to complete DNA replication as replisomes converge has recently been shown to be a highly regulated, multi-enzymatic process. Converging forks also are likely to generate unique supercoiled, tangled, or knotted substrates. These structures are typically resolved by one of the four topoisomerases encoded by Escherichia coli. However, identifying the cellular substrates and specific function for these essential enzymes which contain overlapping biochemical activities has remained challenging. Here, we show that Topo I and Topo IV are required to allow converging forks to complete chromosome replication. Impaired Topo I function leads to amplifications where forks converge, whereas inactivation of Topo IV prevents forks from converging and produces a dramatic loss of this chromosome region. The results are consistent with previous studies suggesting Topo I suppresses illegitimate initiations in the terminus region by disrupting R- and D-loops and demonstrate a specific requirement for Topo IV acting before replication completes to allow convergent forks to reach their doubling point. We propose that the positive supercoils arising between convergent forks are converted to precatenanes and resolved by Topo IV, when diminishing space may preclude gyrase from binding and functioning.