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

Abstract

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 siRNA library covering 240 human DNA damage response (DDR) genes and select siRNAs 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/CHK2 and ATR/CHK1 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 DSBs and post‐replicative ssDNA 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. DNA‐RNA hybrids are a source of spontaneous DNA damage. DNA damage checkpoint (ATR/CHK1, ATM/CHK2 pathways) and post‐replicative repair factors (UBE2B, RAD18) safeguard against DNA‐RNA hybrids at the different stages of the cell cycle. DNA‐RNA hybrids are a natural source of spontaneous DNA damage. The post‐replicative repair and DNA damage checkpoint pathways prevent DNA‐RNA hybrid accumulation by promoting repair of the hybrid‐driven DNA damage. DNA‐RNA hybrids form spontaneously by different mechanisms at each cell cycle stage with distinct impact on DNA replication and the DNA damage response. DNA‐RNA hybrids are a source of spontaneous DNA damage. DNA damage checkpoint (ATR/CHK1, ATM/CHK2 pathways) and post‐replicative repair factors (UBE2B, RAD18) safeguard against DNA‐RNA hybrids at the different stages of the cell cycle.