High-Resolution Mapping of DNA Replication and Double-Strand Breaks in Cancer Cells Reveals Predominant MUS81- and Partial GEN1-Dependence of Recurrent DNA Break Clusters

Genome instability in cancer arises from transcription–replication conflicts and replication stress, yet how these processes shape DNA double-strand break (DSB) landscapes remains unclear. We generated and integrated high-resolution replication timing (HiRepli-Seq), transcription (GRO-seq), and DSB mapping (LAM-HTGTS) in U2-OS and HeLa cells to systematically chart replication stress–induced fragility. Endogenous DSBs localized to origin-rich, R-loop–enriched initiation zones, whereas polymerase inhibition shifted breakage toward late-replicating, origin-poor domains, forming recurrent DNA break clusters (RDCs). RDCs occurred mainly in long, moderately transcribed genes, with transcription modulating their cell type–specificity. Notably, LINGO2 emerged among the most fragile genes under replication stress, frequently accumulating DSBs across cell types. Mechanistically, MUS81 broadly promoted RDC-associated DSBs at both fragile site–linked and non-fragile loci, while GEN1 acted in a more locus-specific manner. These findings define distinct replication- and transcription-driven pathways to genome fragility in cancer cells and highlight the contributions of structure-specific nucleases. Our resource provides a framework for interpreting replication stress, fragile sites, and oncogenic genome evolution.