How Topoisomerases Relax and Disentangle the Genome During DNA Replication

DNA replication generates topological stress that must be relaxed and molecular entanglements that must be resolved. Unwinding of the parental duplex creates positive supercoils ahead of the fork, while fork rotation transfers parental intertwines onto nascent duplexes as precatenanes behind. Unresolved, these structures impede fork progression and prevent sister chromatid separation at mitosis. Cells deploy DNA topoisomerases to manage this topological load, yet which enzymes predominate and what substrates they resolve vary considerably across replication systems. This review examines topoisomerase deployment at canonical replication forks in bacteria, SV40, yeast, and vertebrates. Each system employs distinct strategies for managing topological stress during elongation and termination, reflecting differences in protein-protein interactions, cell cycle control, chromatin structure, and endogenous metabolites. Despite substantial progress, fundamental questions remain unanswered: how cells achieve complete unlinking despite the reversible chemistry of topoisomerases; what determines the supercoil-precatenane balance; how topological stress mechanistically stalls forks; and to what extent replication-specific functions can be disentangled from transcriptional and mitotic roles.