Abstact

Type IIA topoisomerases modulate DNA topology by coordinating the cleavage of gate-segment DNA and the passage of transport-segment DNA-a mechanism conserved across species and essential for diverse cellular processes. While gate-segment interactions have been extensively studied, direct structural evidence of transport-segment capture has remained elusive, limiting our understanding of the full catalytic cycle. Here, we present a cryo-electron microscopy structure of the T4 bacteriophage topoisomerase II with a transport-segment DNA bound directly to its central domain. The structure reveals conformational rearrangements in the central domain that accommodate the transport-segment DNA, suggesting an alternative sequence of events in which the enzyme sliding along the loosely bound religated gate segment may precede transport-segment passage through the coiled-coil gate. Supported by mutational and biochemical assays, our findings provide previously unidentified mechanistic insights and open potential avenues for the development of next-generation type IIA topoisomerase inhibitors.