Topoisomerase IIIα resolves inter- and intra-molecular intertwines during DNA replication

Resolution of topological stress is crucial for genome integrity. Vertebrate topoisomerase II (TOP2) resolves catenanes to relieve topological stress and unlink daughter molecules during DNA replication. Topoisomerase III (TOP3) can also resolve catenanes, but its direct role during DNA replication, substrate specificity, and relevant binding partners remain unclear. Here we show that TOP3 becomes crucial when TOP2 function is compromised. We find that in Xenopus egg extracts, TOP3 promotes replication fork progression and daughter strand unlinking specifically during replication termination. TOP3 can carry out this role independently of its binding partners RMI1-RMI2 and the BLM helicase by acting on lagging-strand single-stranded DNA (ssDNA). Strikingly, elevated lagging-strand ssDNA drives formation of intramolecular ssDNA intertwines, which are ordinarily resolved by TOP3. Thus, TOP3 resolves intermolecular linkages to promote fork progression during termination and resolves intramolecular linkages that arise when high levels of ssDNA are present during DNA replication.