Linear interaction between transcription and replication shapes DNA break dynamics

Recurrent DNA break clusters (RDCs) are replication and transcription collision hotspots. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication fork architecture dictating RDC location and orientation. Most RDC occurs at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSB) while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bounded RNA revealed transient DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping nascent RNA and RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery slow down DNA replication, resulting in 60% DSB contribution to the head-on as compared to 60% for co-directional . Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability. Overall design: We performed high-throughput sequencing to measure the linear movement of transcription and DNA replication in mouse neural progenitor cells.