Genomewide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression

DNA replication progression can be affected by the presence of physical barriers on the DNA, like the RNA Polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know the overall influence of transcription on DNA replication progression. To characterize what happens at sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints to identify replication fork/stalling hotspots as the replication progresses through the genome. These sites are typically associated with increased DNA damage, overlap with fragile sites and with breakpoints of rearrangements identified in cancers, but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA Polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. We also find instances where transcription activity favors replication progression because reduces histone density, but also that slowing down transcription elongation slows down directly replication progression through genes. This would indicate that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.