Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Significance R-loop formation has been related to genome instability and human disease, yet the role of R-loops in replication priming remains to be explored in the eukaryotic genome. This investigation discloses that transcription-dependent R-loops have the potential to initiate origin-independent replication events in ribosomal DNA. Taken together, our data suggest that R-loops contribute to transcription-driven endoreplication events and alterations in genome copy number. DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop–mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis.