Transcription–replication collisions trigger high-fidelity replication reset

Double-stranded DNA ends arise from external agents or cellular processes like transcription-replication collisions (TRCs), threatening genome stability. Here, we performed genomic CRISPRi screens to uncover DNA end formation factors in Escherichia coli. We discovered that translation-transcription decoupling causes DNA end formation through a TRC-dependent pathway, which is lethal when DNA end processing by RecBCD is disrupted, but not when recombination is disrupted. We find that TRCs cause replisome stalling followed by "rear-ending" from trailing replisomes which generates free DNA ends, rather than strand breaks. Surprisingly, these DNA ends are resolved through a process we call "replication reset", where the stalled replicore is degraded, without triggering recombination, the DNA damage response, or mutagenesis. This hidden replicore-degradation resets the replication cycle without consequence for the genome. This discovery reveals a novel DNA safeguard mechanism for preserving genome stability when replication is disturbed and challenges the notion that TRCs necessarily cause genome instability in bacteria.