DNA topoisomerases have an important function in preventing replication/transcription (R/T) conflicts caused by RNA polymerases elongating in the opposite (or same) direction of advancing replication fork. Published data demonstrated that such persistent R/T conflicts can be rescued by overexpression of RNase H1, which specifically degrades the RNA of DNA:RNA hybrids present in R-loop structures. R loops are a common non-B DNA structure of cellular genomes that mainly occur during transcription. R loops can lead to genome instability; however, they also play a role in important cellular functions. Here, we have determined the dynamics of cellular R loops generated by chemical poisoning of DNA topoisomerases (TOP1 and TOP2) and the role of the proteasome in DNA damage induction by performing detailed kinetics analyses of cellular R loops by immunofluorescence microscopy. Treatment of HeLa and U2OS cells with clinically used topoisomerase inhibitors (camptothecin, LMP-776, MJ-III-65, doxorubicin, sabarubicin) triggers an immediate increase of nucleoplasmic R loops from 2 to 10-20 minutes of treatment, followed by a dramatic reduction of R-loop levels at 1 hour. Concomitantly with the reduction of R-loop structures, DNA damage was detected as phosphorylation of histone H2AX, a marker of DNA double-stranded breaks (DSB). Pretreating cells with MG132 (a proteasome inhibitor) stabilized the presence of R loops at longer treatment times and concomitantly reduced DSB formation, suggesting that the degradation of poisoned topoisomerases is necessary to trigger the pathway resulting in DSB formation. Overexpression of RNaseH1 in U2OS cells abolishes the transient increase of R loops and the induction of DNA damage. Topoisomerase poisoning is able to effectively kill the studied human cancer cells. However, overexpression of RNaseH1 or MG132 markedly reduced cell killing induced by R/T conflicts generated by topoisomerase inhibitors. The results are overall consistent with a necessary function of nuclear proteasome in generating a free DNA break from abortive topoisomerase cleavage complexes that will eventually lead to DSBs.
Citation Format: Jessica Marinello, Maria Delcuratolo, Yves Pommier, Monica Binaschi, Andrea Pellacani, Giovanni Capranico. DNA topoisomerase-mediated transcription-replication conflicts cause DNA damage by a transient increase of R loops and proteasome activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B188.