Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, and evolution across species. Head-on conflicts (lagging-strand genes) are significantly more detrimental than codirectional conflicts (leading-strand genes). The fundamental reason for this difference is unknown. Here, we report that topological stress significantly contributes to this difference. We find that head-on, but not codirectional, conflict resolution requires the relaxation of positive supercoils by the type II topoisomerases DNA gyrase and Topo IV, at least in the Gram-positive model bacterium Bacillus subtilis. Interestingly, our data suggest that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. Altogether, our results reveal a fundamental mechanistic difference between the two types of encounters, addressing a long-standing question in the field of replication-transcription conflicts.