biostudies-literatureWesthorpe RMedical Research Council3469-3481.e7https://www.ebi.ac.uk/biostudies/studies/S-EPMC7617106biostudies-literatureUnknown84(18)Topoisomerase 1 cleavage complexes (Top1-ccs) comprise a DNA-protein crosslink and a single-stranded DNA break that can significantly impact the DNA replication machinery (replisome). Consequently, inhibitors that trap Top1-ccs are used extensively in research and clinical settings to generate DNA replication stress, yet how the replisome responds upon collision with a Top1-cc remains obscure. By reconstituting collisions between budding yeast replisomes, assembled from purified proteins, and site-specific Top1-ccs, we have uncovered mechanisms underlying replication fork stalling and collapse. We find that stalled replication forks are surprisingly stable and that their stability is influenced by the template strand that Top1 is crosslinked to, the fork protection complex proteins Tof1-CsMolecular cellMechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes.PMC7617106MC_UP_1201/12Roske JJWesthorpe RYeeles JTPfalseMechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes.Topoisomerase 1 cleavage complexes (Top1-ccs) comprise a DNA-protein crosslink and a single-stranded DNA break that can significantly impact the DNA replication machinery (replisome). Consequently, inhibitors that trap Top1-ccs are used extensively in research and clinical settings to generate DNA replication stress, yet how the replisome responds upon collision with a Top1-cc remains obscure. By reconstituting collisions between budding yeast replisomes, assembled from purified proteins, and site-specific Top1-ccs, we have uncovered mechanisms underlying replication fork stalling and collapse. We find that stalled replication forks are surprisingly stable and that their stability is influenced by the template strand that Top1 is crosslinked to, the fork protection complex proteins Tof1-Cs2024-01-01T00:00:00Z2024 Sep2025-04-03T23:56:16.387Z2025-04-03T23:56:16.387ZS-EPMC76171063923671910.1016/j.molcel.2024.08.004