Endogenous DNA damage at sites of terminated transcripts

DNA damage promotes mutations that fuel cancer, ageing and neurodegenerative diseases^{1, 2–3}, but surprisingly, the causes and types of damage remain largely unknown. There are three identified mechanisms that damage DNA during transcription: collision of RNA polymerase (RNAP) with the DNA-replication machinery head-on and co-directionally^{4, 5–6}, and R-loop-induced DNA breakage^{7, 8, 9–10}. Here we identify novel DNA damage reaction intermediates^11,12 and uncover a fourth transcription-related source of DNA damage: endogenous DNA damage at sites of terminated transcripts. We engineered proteins to capture single-stranded DNA (ssDNA) ends with 3′ polarity in bacterial and human cells. In Escherichia coli, spontaneous 3′-ssDNA-end foci were unexpectedly frequent, at one or more per cell division, and arose via two identifiable pathways, both of which were dependent on DNA replication. A pathway associated with double-strand breaks was suppressed by overexpression of replicative DNA polymerase (pol) III, suggesting competition between pol III and DNA damage-promoting proteins. Mapping of recurrent 3′-ssDNA-ends identified distinct 3′-ssDNA-end-hotspots, mostly unrelated to double-strand breaks, next to the 5′-CCTTTTTT transcription-terminator-like sequence. These 3′-ssDNA-termini coincide with RNA 3′-termini identified by DirectRNA sequencing¹³ or simultaneous 5′ and 3′ end RNA sequencing (SEnd-seq)¹⁴ and were prevented by a mutant RNAP that reads through terminators. Our findings reveal that transcription termination or pausing can promote DNA damage and subsequent genomic instability.