Replication Stress caused by Post-replicative RNA:DNA hybrids – ReSPoND

R-loops are natural by-products of transcription and prevalent three-stranded nucleic acid structures consisting of an RNA:DNA hybrid and an unpaired DNA strand. They play a key role in many critical physiological processes, such as mitochondrial replication, class-switch recombination at immunoglobulin genes, or regulation of gene expression. However, when their turnover is affected or when cells experience a DNA replication stress, some R-loops become problematic and severely compromise genome integrity. Despite an intense scrutiny, the mechanisms involved remain elusive. Our ground-breaking and far-reaching preliminary data suggest a new working hypothesis in which pre-existing R-loops are transferred behind replication forks by the dissociation of the leading and lagging strand machineries, where they interfere with normal fork restart. Our working model makes strong predictions that we aim to test directly in this proposal. Importantly, we have already set up an innovative toolkit that allows us for the first time to rapidly manipulate R-loops in vivo in human cells, specifically during DNA replication stress. This new tool puts this consortium in a unique position to address our working hypothesis and will be of paramount importance to the success of this project.

We have three main objectives in this project: (i) use four independent but complementary approaches to generate exhaustive maps of R-loop forming loci upon replication stress in human cells, distinguishing R-loops that form in front of replication forks from R-loops that form behind forks, hereafter termed post-replicative hybrids. Based on our previous data, we expect to detect such problematic post-replicative hybrids near the 3’ ends of highly transcribed genes; (ii) demonstrate that post-replicative hybrids arise from pre-existing R-loops; and finally (iii) understand how post-replicative hybrids interfere with fork restart. This proposal is a direct continuation of the ReDeFINe project previously funded by the ANR, in which the three partners of the current proposal were already involved. They have complementary expertise and are used to working together, which will be a significant asset for the current proposal.