PROJECT SUMMARY Replication and transcription are fundamental physiological processes, yet paradoxically they both threaten genome stability. Replication forks encounter various types of endogenous and exogenous obstacles that keep them from accurately completing DNA replication. Recent studies suggest that the deleterious effects of transcription could be a consequence of R-loops, three- stranded nucleic acid structures containing an RNA-DNA hybrid and a region of single-stranded DNA. R-loops occur throughout the genome of mammalian cells and regulate various aspects of gene expression, but their accumulation leads to DNA damage, particularly when cells are undergoing DNA replication. Increasing evidence suggests that conflicts between transcription- associated R-loops and replication protein complexes are important factors underlying genome instability, but the specific mechanisms driving this instability are currently unknown. The long- term goal of this research program is to understand how cells distinguish and resolve regulatory and deleterious R-loops, and how this is perturbed in human disease. It is hypothesized that R- loops are dynamic structures that become susceptible to processing when they accumulate, leading to the formation of DNA breaks and ultimately resulting in genome instability. The object of this application is to define how R-loops are recognized and processed in the cells throughout the cell cycle, to determine where in the genome processing occurs, and to determine how conflicts with the replication machinery contribute to R-loop processing. In the first aim, the processing of R-loops by cellular endonucleases involved in DNA repair will be explored in cells using molecular and cell biological approaches. In the second aim, the sites and products of R- loop formation and processing will be identified and mapped. These studies will take advantage of cutting-edge genomic approaches that have been developed to map the spatial distribution of R-loops and R-loop processing products throughout the genome. Finally, in the third aim, the impact of an R-loop on collisions between replication and transcription machineries will be studied in cells. These studies will take advantage of a novel system recently developed to control such collisions and R-loop formation in the context of the replication fork and recent break-mapping strategies.