Endogenous DNA damage at sites of terminated transcripts

DNA damage can lead to mutations that fuel cancer, aging, and neurodegenerative diseases, but surprisingly, the causes and types of damage remain largely unknown. There are three identified mechanisms that damage DNA during transcription: RNA polymerase (RNAP) colliding with DNA-replication machinery head-on and co-directionally, and R-loop-induced DNA breakage. Here, we identify DNA-damage reaction intermediates uncharacterized previously in living cells, and uncover a surprising fourth transcription-related source: endogenous DNA damage at transcription-termination sites. We engineered proteins to capture single-stranded (ss)DNA ends with 3'-polarity, in both 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 dependent on DNA replication. A pathway associated with double-strand breaks (DSBs) was suppressed by overproduction of replicative DNA polymerase (pol) III, suggesting competition between pol III and DNA-damage-promoting proteins. We developed ThreeSSeq, genomic profiling of 3'-ssDNA-ends, and found distinct 3'-ssEnd-hotspots, mostly unrelated to DSBs, next to the 5'CCTTTTTT transcription-terminator-like sequence. These 3'-ssDNA-termini coincide with RNA 3'-termini identified by DirectRNA sequencing or SEnd-seq and were prevented by a mutant RNAP that reads-through terminators. Our findings reveal that transcription termination or pausing can promote endogenous DNA damage and subsequent genomic instability.