Release of Paused Pol II Induces Double-Strand Breaks in Normal Cells

DNA double-strand breaks (DSB) can occur as a result of various external genotoxic stresses or cell-intrinsic processes, and misrepair of DSBs can contribute to aberrant mutations or translocations. Persistent DSBs are also common in normal, unstressed cells and have been associated with transcriptional activity, but how these spontaneous DSBs form and resolve remains unclear. Dellino, Palluzzi, and colleagues show that DSBs accumulate in normal cells following release of paused RNA polymerase II (Pol II) at discrete genomic sites and that these breaks are associated with sites prone to chromosomal translocation. In normal proliferating mammary epithelial cells, endogenous DSBs were significantly enriched at gene promoters, intron 5′ splice sites, and active enhancers. Fragile (DSB-positive) promoters exhibited paused Pol II phosphorylated at serine 5 of the carboxy-terminal domain (Pol II-pS5) and were found at genes longer than undamaged control genes with comparable transcription levels; fragile introns were similarly longer than controls. Fragile promoters were also bound by topoisomerase 2B (TOP2B) and the DNA-repair protein XRCC4, which correlated with levels of paused Pol II-pS5, suggesting that topoisomerase activity contributes to endogenous DSB formation and that these DSBs are processed by end-joining DNA-repair pathways. Inhibition of transcriptional elongation resulted in loss of XRCC4 at fragile promoters and 5′ splice sites, whereas removal of the inhibitor and release of paused Pol II resulted in increased binding of XRCC4, suggesting that release of Pol II pausing is a primary factor in DSB formation at these sites. Of note, fragile introns were highly enriched for translocation breakpoints when considering those mapping to boundary-flanking regions of topologically associating domains that showed high interaction frequencies with distal loci. Taken together, these findings indicate that release of paused Pol II at specific genomic loci and chromatin domains allows for DSB formation in normal cells and can contribute to cancer-associated chromosomal translocations.

Dellino GI, Palluzzi F, Chiariello AM, Piccioni R, Bianco S, Furia L, et al. Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations. Nat Genet 2019 May 20 [Epub ahead of print].

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