BRCA2 suppresses “head-on” transcription-replication conflicts incurred by dormant origin firing at transcription termination sites [Ok-seq]
BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from genotoxicity and replication stress, but the mechanism(s) by which this is achieved remains elusive. Here, we provide evidence that BRCA2 acts as a critical suppressor of “head-on” transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified new origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNaseH2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results identify a new source of genomic instability caused by HO-TRCs in BRCA2-deficient ovarian cancer precursor cells, providing a new strategy in modulating HO-TRCs to enhance genomic instability Overall design: Okazaki-fragment (Ok-seq) sequencing was performed on fallopian tube epithelial cells (FTEs) with a doxycyline (DOX)-inducible shRNA against BRCA2 after 0 days (untreated), 3 days, and 6 days of BRCA2-knockdown. Two replicates of Ok-seq were performed per sample.