R-loops and Topoisomerase 1 facilitate DSBs formation at gene body of actively transcribed genes

DNA double-stranded breaks (DSBs) are considered to be the most deleterious type of DNA damage. Although cytotoxic levels of DSBs occur mainly due to external factors such as ionizing irradiation and UV radiation, DSBs can occur naturally under physiological conditions through cell’s natural processes such as transcription and replication. Transcription can cause DSBs in replicating cells by colliding with replication, especially when transcription and replication have opposite directions. However, how physiological DSBs are generated during transcription is still poorly understood. Here, we mapped DSBs using in-suspension break labeling in situ and sequencing (sBLISS) method and re-analyzed ChIP-seq and DRIP-seq data, along with immunofluorescence staining for DNA damage markers in MCF-7 and U2OS cells. Mapping of DSBs revealed their enrichment at highly expressed genes which are coupled with high Topoisomerase 1 (Top1), the main enzyme responsible for resolving positive and negative supercoiling, and R-loops levels. Depleting R-loops and Top1 decreased DSBs specifically at high expression/Top1/R-loops genes implying that Top1 and R-loops are the main causes of transcriptional DSBs. These findings propose a model in which DSBs in oncogenes are mainly caused by the accumulation of R-loops and the catalytic activity of Top1. Our data also reveal that these oncogenes, despite being highly fragile, are also highly expressed, and this high expression is mediated by efficient repair signaling as indicated by γ-H2AX accumulation. Our findings underscore the critical roles of Top1 and R-loops in regulating hypertranscription of oncogenes implicated in carcinogenesis.