Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, a unique agent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides new therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, a full mechanistic understanding of the drug's TC-NER-dependent toxicity is needed. Here, we determined that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) by blocking the second of the two sequential NER incisions by XPG. We mapped the 3'-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. We showed that trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs were also found outside gene bodies, revealing TC-NER connection to divergent transcription from promoters. This work advances trabectedin as a tool compound for precision oncology and for studying TC-NER and transcription. Overall design: Genome-wide mapping of DNA breaks with GLOE-seq in U2OS WT, CSB knockout (-KO), XPC-KO and XPA-KO cells as well as in HAP1 WT cells that were exposed to trabectedin (50 or 20 nM) or DMSO (as a control) in 1 to 3 biological replicates per exposure-cell line combination. In addition, genome-wide mapping of DNA breaks with GLOE-seq in U2OS WT gDNA treated with the nicking endonuclease Nb.BsrDI.