RNA:DNA hybrids and replication fork instability – ReDeFINe

R-loops are stable structures that form during transcription when the nascent RNA reanneals with its template DNA, generating an RNA:DNA hybrid and a displaced ssDNA loop. These structures cover up to 5% of the human genome and play pivotal functions in the regulation of gene expression. A large body of evidence indicates that R-loops represent also a potential source of genomic instability, most likely by increasing transcription-replication conflicts (TRCs) in an orientation-dependent manner. R-loops differ by their length, sequence and by the genomic context in which they assemble. However, the mechanism by which R-loops interfere with DNA replication remains poorly understood. Preliminary evidence indicates that RNA:DNA hybrids also form at arrested forks and that they interfere with the restart of stalled forks if they are not readily removed. Together, these data indicate that RNA:DNA hybrids represent not only a cause, but also a consequence of replication stress (RS), with deleterious consequences for the integrity of the genome. The aim of the ReDeFINe project is to characterize this dual impact of RNA:DNA hybrids by addressing three fundamental questions:
- Are all R-loops toxic for replication forks and what determines their toxicity?
- Do RNA:DNA hybrids assemble at stalled forks as a consequence of replication stress?
- Do RNA:DNA hybrids interfere with the restart of stalled forks?
The ReDeFINe project will tackle these critical questions by addressing the distribution of toxic RNA:DNA hybrids and their impact on fork arrest/restart in three model organisms (S. cerevisiae, S. pombe and human cell lines). This ambitious project will be conducted by a consortium of four groups with expertise in DNA replication (Pasero, Lambert and Chen), R-loop mapping (Vanoosthuyse, Pasero) and bioinformatics (Chen). The use of three complementary model organisms will reveal universal mechanisms linking RNA:DNA hybrids to replication-associated genome instability. The consortium will exploit state-of-the-art assays to monitor fork arrest at the genome-wide level and at specific loci. It will generate large genomic datasets and will combine them to generate the first comprehensive genomic landscape of R-loops and replication stress markers in yeasts and human cells. These maps will allow us to distinguish between co-transcriptional R-loops that interfere with fork progression from those that form as a consequence of DNA replication stress. They will also be used to identify features that determine the toxicity of co-transcriptional R-loops under normal growth conditions. Finally, the ReDeFINe project will determine the nature of RNA:DNA hybrids that accumulate at stalled forks and will characterize their impact on fork processing and restart in model organisms and in the context of human diseases. Altogether, these studies will shed new light on mechanisms by which R-loops induce genomic instability during DNA replication in yeasts and human cells.