Project Summary Maintenance of genomic integrity depends on the ability of cells to repair damaged DNA and resolve transcription-replication conflicts. In this regard, R-loops, three-stranded nucleic acid structures that harbor an RNA transcript hybridized to a DNA template, can compromise genome stability in multiple ways. Specifically, the ssDNA within the R-loop structure is vulnerable to nucleolytic cleavage, resulting in transcription-associated mutagenesis or transcription-associated recombination. Moreover, collisions of the DNA replication machinery with R-loops can cause replication fork collapse, DNA double-strand breaks (DSBs), fork fusions, and chromosome translocations, which can then lead to neoplastic transformation and tumorigenesis. This competitive continuation of our MPI grant leverages our unique expertise in DNA repair enzymology and cell biology modeling to delineate the structure-function of an R-loop resolution machinery comprised of the SF1 family helicase Senataxin (SETX) and the tumor suppressor complex BRCA1-BARD1. In Specific Aim 1, we will define the unusually versatile nucleic acid unwinding activity of SETX and test the hypothesis that SETX resolves R loops directly through specific unwinding activity. Specific Aim 2 will determine the role of BRCA1-BARD1 in SETX-mediated R-loop resolution to test the hypothesis that BRCA1-BARD1 cooperates with SETX to resolve pathological R-loops by interrogating SETX and BRCA1-BARD1 in our reconstituted biochemical systems and in cells. This MPI renewal is based on the longstanding and productive collaboration between Dr Patrick Sung, a leading DNA repair enzymologist, and Dr Gary Kupfer, a physician-scientist who has utilized the genetic model of Fanconi anemia to advance understanding of DNA repair pathways and mechanisms. Together, with numerous coauthored papers of high significance, our continuing collaborative endeavors promise to exert impact of the highest degree and to provide insight into the mechanistic underpinnings of a major genome maintenance pathway that is linked to tumor suppression pathways.