Topoisomerase I poisoning induces R-loop changes and DNA damage in human cancer cells [DRIP-seq]

DNA topoisomerase I is an essential enzyme in higher eukaryotes that regulates DNA torsional tension during fundamental processes, such as replication and transcription. During its catalytic activity, a transient Topoisomerase I-DNA cleavage complex, called Top1cc, forms to allow strand rotation and duplex relaxation. However, the stabilization of Top1cc can lead to increased DNA:RNA hybrid duplexes, DNA double-strand cuts (DSBs) and genome instability as shown by formation of micronuclei, extranuclear chromatin enveloped by a nuclear membrane. To better comprehend the underlying mechanisms, we have established genomic maps of Top1cc-mediated R-loop changes and DSBs at short times upon Top1cc induction. Our findings show that Top1ccs dynamically changed the genomic distribution of R-loops, with regions of stable hybrid gains. DSBs were at specific gene loci, strongly associated with stable anterior R-loops at highly-transcribed genes and close to backtracked RNA polymerase II. Moreover, DSBs and stable anterior R-loops were highly enriched at early replication origins, thus revealing the location of replication conflicts with backtracked RNA polymerases. DRIP-seq experiments were performed on HCT116 cells treated with Camptothecin (CPT) for 5 and 60 minutes and in untreated cells. For each DRIP library a library on RnaseH-treated sample was produced to filter false positive DRIP regions. We produced 2 biological replicates per condition.