The DNA damage response acts as a safeguard against harmful DNA–RNA hybrids of different origins

Despite playing physiological roles in specific situations, DNA – RNA hybrids threat genome integrity. To investigate how cells do counteract spontaneous DNA – RNA hybrids, here we screen an si RNA library covering 240 human DNA damage response ( DDR ) genes and select si RNA s causing DNA – RNA hybrid accumulation and a significant increase in hybrid‐dependent DNA breakage. We identify post‐replicative repair and DNA damage checkpoint factors, including those of the ATM / CHK 2 and ATR / CHK 1 pathways. Thus, spontaneous DNA – RNA hybrids are likely a major source of replication stress, but they can also accumulate and menace genome integrity as a consequence of unrepaired DSB s and post‐replicative ss DNA gaps in normal cells. We show that DNA – RNA hybrid accumulation correlates with increased DNA damage and chromatin compaction marks. Our results suggest that different mechanisms can lead to DNA – RNA hybrids with distinct consequences for replication and DNA dynamics at each cell cycle stage and support the conclusion that DNA – RNA hybrids are a common source of spontaneous DNA damage that remains unsolved under a deficient DDR .