Mapping of physiological DNA double stranded breaks in normal breast cells and breast cancer cells [BLISS]
DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employed the advanced BLISS techniques to map DSBs, with different genetic manipulations to comprehensively investigate the interplay between transcription, DSBs, Topoisomerase 1 (TOP1), and R-loops. Our findings revealed the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops, indicating their crucial involvement in transcription-associated genomic instability. Depletion of R-loops and TOP1 specifically reduced DSBs at highly expressed genes, uncovering their pivotal roles in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides novel insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development. Notably, our study highlights the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations, shedding light on the potential for targeted therapeutic strategies. Overall, these findings deepen our understanding of the regulatory mechanisms governing DSBs in hypertranscribed genes associated with carcinogenesis, opening avenues for future research and therapeutic interventions. Overall design: In-suspension break labeling in situ and sequencing (sBLISS) for MCF-7 cells, for control cells transfected with scramble siRNA and invected with empty vector (EV), cells after TOP1 knockdown (KD), cells after RNase H overexpression (OE), cells after both manipulations (TOP1 KD + RNase H OE). sBLISS was also performed on MCF-7 cells after E2 treatment, E2 treatment and RNase H OE, and control cells treated with vehicle and infected with EV. Additionally sBLISS was also performed on MCF-7 cells after E2 treatment, E2 treatment and TOP1 KD, and control cells treated with vehicle and transfected with scrambleRNA. sBLISS was also performed on synchronized MCF-7 cells in the G1 cell cycle stage. sBLISS was also performed on HMLE cells, and MCF-10A WT cells as well as MCF-10A RAS transformed cells. all BLISS experiments were replicated in duplicates, and in quadruplicates in HMLE cells.