DNA Replication Modulates R-loop Levels to Maintain Genome Stability

Conflicts between transcription and replication are a potent source of DNA damage. The transcription machinery has the potential to aggravate such conflicts by generating co-transcriptional R-loops as an additional barrier to replication fork progression. Here, we investigate the influence of conflict orientation and R-loop formation on genome stability in human cells using a defined episomal system. This approach reveals that head-on (HO) and co-directional (CD) conflicts induce distinct DNA damage responses. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the CD orientation but promoting their formation in the HO orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings not only uncover an intrinsic function of the replisome in R-loop homeostasis, but also suggest a mechanistic basis for genome instability associated with deregulated DNA replication, which is observed in many disease states, including cancer.