R-loops and Topoisomerase 1 facilitate formation of transcriptional DSBs at gene bodies of hypertranscribed cancer genes

Abstract

DNA double-stranded breaks (DSBs) are considered 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. 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) and re-analysis of ChIP-seq and DRIP-seq data, along with immunofluorescence staining for DNA damage markers in MCF-7 cells. Mapping of DSBs revealed their occurrence at highly expressed genes that are enriched with Topoisomerase 1 (Top1), the main enzyme responsible for resolving positive and negative supercoiling, and with R-loops. Depleting R-loops and Top1 decreased DSBs specifically at highly expressed and Top1/R-loops-enriched genes implying that Top1 and R-loops are the main causes of transcriptional DSBs. These findings propose a model in which DSBs in highly expressed genes, mainly oncogenes, are caused by resolving of R-loops and the catalytic activity of Top1. Our data also reveal that these genes, despite being highly fragile, are covered by γ-H2AX suggesting efficient repair signaling. Our findings underscore the critical roles of Top1 and R-loops in regulating DSBs in hypertranscribed oncogenes implicated in carcinogenesis.