Harmful R loops are prevented via different cell cycle specific mechanisms

R loops induce transcription-replication (T-R) conflicts, but recent reports have suggested that R-loops are caused by T-R conflicts. Identifying how R loops are generated is crucial to know how transcription compromise genome integrity. Genome-wide analyses of conditional yeast mutants reveal that transcription factors, such as the THO complex. prevents R loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the RF in sen1 cells but symmetrically in the hpr1 THO mutant. Our results unambiguously indicate that: R loops forms co-transcriptionally independently of DNA replication; THO is a general and cell-cycle independent safeguard against R loops, and Sen1, in contrast to previously believed, is an S-phase-specific R loop resolvase. These conclusions have important implications for the mechanism of R loop formation and the role of other factors reported to impact on R loop homeostasis DNA-RNA immunoprecipitation strand-specific (DRIPc-seq) were performed on WT YMK612 and hpr1 and sen1 depleted cells from G1-synchronized or S-phase cultures, treated or not with Rnase H. H2AP ChIP-seq were performed on WT YMK612 and hpr1 and sen1 depleted cells from S-phase cultures.