Transcription-Replication Collision Shapes DNA Break Dynamics

Transcription and replication conflict (TRC) are one of the main driving forces for genome instability. Yet, TRC rarely been discussed without the context of DNA:RNA entanglement, rending the role of transcription in other TRC unclear . In neural stem and progenitor cells, genes encode protein regulating neuron adhesion are hotspots for recurrent DNA break clusters (RDC). While RDC-containing genes are all actively transcribed, most RDC lack DNA:RNA entanglement. We demonstrated that, through controlled gain and loss of function genetic approaches, transcription activity is essential while not sufficient to induce RDC formation. In combination of a deep neural network and single-nucleotide resolution DNA break mapping approaches, we found RDC break densities mirror the replication fork dynamics. We demonstrated that, for the first time that, head-on TRC results in higher DNA break density than its co-direction counterparts. In summary, our results revealed that transcription has a higher-level regulatory role that has to be coordinated with DNA replication. Overall design: Two parental lines of mouse ES cell (NXP010, NXP047) were used for CRISPR-Cas genome editing to create daughter lines that lack one allele of Ctnna2 (47-5_Ctnna2_allele-B), Ctnna2 promoter-enhanced deleted ES cell clones (38-3_Ctnna2_allele-B_promoter-A and 18-4_Ctnna2_allele-B_promoter-A), or ES cell lines that lacks one allele of Nrxn1 (22_Nrxn1_allele-B), and ), and Nrxn1 promoter-enhanced deleted ES cell clones (22-5_Nrxn1_allele-B_promoter-A and 22-37_Nrxn1_allele-B_promoter-A). The parental ES cells and the derived ES cell clones were in vitro differentiated to neural progenitor cells (ESC-NPC). These ESC-NPC were used for nascent RNA analyses, the Global-run on Sequencing (GRO-seq). To determine early and late replciation profile, we performed two fraction Repli-seq to ESC-NPC lines that lack one allele of Ctnna2 or Nrxn1, and lines that removed the promoter-enhancer of Ctnna2 or Nrxn1. For mapping DNA breaks in aphidicolin treated ESC-NPCs, cells were treated with 0.3 uM aphidocolin with or without 5 nM ATR inhibitor VE-821 for 96 hours. For LAM-HTGTS in ESC-NPCs, we used Chr6 and Chr8 viewpoints (Chr6_70Mb, Chr8_61Mb). For mapping DNA breaks in the CAST/121-9 ES cells, we used an ES cell line that the Ptn promoter on the 121-9 allele is replaced with an tetracyclin reponding element. The ES cells were treated with or without 0.1 uM aphidicolin. To induce Ptn expression, cells were treated with 0.125, 0.25, or 0.5 uM tetracycline. Viewpoint on Chr6 (Chr6_70Mb) was used in the CAST/121-9 experiments.