PROJECT SUMMARY Maintaining genome integrity is paramount for all cells, yet the essential cellular process of gene transcription introduces myriad opportunities for cells to incur damage to their genome. For example, transcription can lead to the formation of R-loops, three-stranded structures that contain 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. They are typically dynamic, but some sequences and cellular perturbations can lead to stable R-loops that can stall transcription, block DNA replication, or cause DNA breaks via incompletely understood mechanisms. Moreover, some of these R-loops can be excised from the genome and accumulate in the cyto- plasm, where they can trigger innate immune responses and even cell death. Thus, cells carefully regulate R- loop formation and turnover. The long-term goal of this research program is to understand how cells identify deleterious R-loops (as dis- tinct from regulatory ones), how problematic R-loops are removed from the genome, and how these functions are disrupted in human diseases. Data suggest that stabilized R-loops become susceptible to processing (i.e., excision from the genome, followed by gap repair) when less mutagenic pathways such as unwinding of the RNA-DNA hybrid or degradation of the RNA component on the chromosome are unavailable or too slow. The objective of this application is to identify the pathway(s) that excises problematic R-loops from the genome and to define the sites and sequence characteristics of problematic R-loops. Aim 1 will investigate which DNA repair factors are involved in R-loop processing and determine how the nucleases that process these structures are recruited to the sites where R-loops are excised. Studies are also proposed to determine whether R-loop excision leaves a single-stranded gap in the DNA, to define the sites of nuclease processing and gap formation, and to identify the polymerases responsible for repairing these gaps. Aim 2 will characterize the genomic and sequence features of problematic R-loops and develop a novel, modu- lar experimental system to directly test hypotheses about the determinants of R-loop processing. These stud- ies will take advantage of new cell biological methods that have been developed to study R-loop processing and cutting-edge genomic approaches developed to map the spatial distribution of R-loops and R-loop pro- cessing products throughout the genome. Elucidating the features of problematic R-loops and how the cell copes with them has the potential to illumi- nate our understanding of how cells maintain genome stability and how this may go awry in cancer and other diseases states. This will lay a strong foundation for strategies to manipulate R-loop processing for therapeutic ends.