Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

SummaryBET bromodomain proteins are required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on processes such as DNA replication will be important for future clinical applications. Here, we show that BET inhibition, and specifically inhibition of BRD4, causes replication stress through a rapid overall increase in RNA synthesis. We provide evidence that BET inhibition acts by releasing P-TEFb from its inhibitor HEXIM1, promoting interference between transcription and replication. Unusually, these transcription-replication conflicts do not activate the ATM/ATR-dependent DNA damage response but recruit the homologous recombination factor RAD51. Both HEXIM1 and RAD51 promote BET inhibitor-induced fork slowing but also prevent a DNA damage response. Our data suggest that BET inhibitors slow replication through concerted action of transcription and recombination machineries and shed light on the importance of replication stress in the action of this class of experimental cancer drugs.

Hypertranscription is defined as a relative increase in global transcription to support proliferation and cell growth.

Obstacles on the DNA template can lead to DNA replication fork stalling and genomic rearrangements. RAD51-mediated homologous recombination (HR) can promote restart and repair of stalled forks, but also post-replicative repair once the obstacle has been bypassed. Bulky DNA adducts are important replication-blocking lesions induced by environmental carcinogens, but it is not known whether they activate HR directly at stalled forks, or at gaps left behind ongoing forks. Here we show that in mammalian cells, bulky adducts predominantly induce HR at post-replicative gaps formed by the DNA/RNA primase PrimPol. Using BPDE and UV model lesions, we report that RAD51 is not recruited to stalled or collapsed forks, but instead to long gaps formed by PrimPol re-priming activity and resection by MRE11 and EXO1. In contrast, RAD51 loading at DSBs does not require PrimPol. At bulky adducts, PrimPol is required for the induction of sister chromatid exchanges and genetic recombination. Our data support that HR at bulky adducts in mammalian cells is primarily associated with post-replicative gap repair and define a role for PrimPol in DNA damage tolerance by homologous recombination.

Highlights d BET inhibitors and BRD4 depletion increase overall RNA synthesis d Loss of BRD4 activity causes conflicts between transcription and DNA replication d These transcription-replication conflicts depend on P-TEFb inhibitor HEXIM1 d BET inhibition activates RAD51 to slow replication forks and suppress DNA damage