Transcription-Replication Conflict Shapes DNA Break Dynamics.

Double strand breaks (DSBs) originating from transcription and replication conflict (TRC) sites are prone to rearrangements. Through the application of a capture-ligation assay on mouse neural progenitor cells experiencing replication stress, we unveiled that interactions between transcription and replication fork architecture dictate DSB location and orientation. Specifically, telomere-to-centromere forks generate telomere-connected DSBs, while centromere-to-telomere forks lead to centromere-connected DSBs in genomes that replicate during median and late stages. This pattern, however, reverses in early-replicating DNA. Mapping nascent RNA and RNA polymerase activity revealed that head-to-head interactions between replication and transcription machineries elevate DSBs by 30% compared to co-directional interactions. By deleting promoter elements in the moderately transcribed Ctnna2 and Nrxn1 genes, we demonstrated that the removal of transcription suppressed DSB. However, titration of expression at the Ptn locus demonstrated that transcription level alone was insufficient to induce DSB. Our findings underscore how transcription and replication directionality impact genome structural stability.