Topoisomerase 1-dependent R-loop deficiency drives accelerated replication and genomic instability

DNA replication is a complex process tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here, we show that mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression, causing aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results dissect a mechanism of oncogene-induced DNA damage by aberrant replication fork acceleration. [Display omitted] •Increased TOP1 expression by mutated RAS reduces R loops•Low R-loop levels promote accelerated replication and DNA damage•TOP1 restoration or mild replication inhibition rescue DNA acceleration and damage•High TOP1 expression is associated with replication mutagenesis in cancer TOP1 deficiency increases R loops driving replication stress characterized by slow replication and DNA damage. Sarni et al. reveal that HRAS increases TOP1 and reduces R loops, causing accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful replication and genome integrity.