Project Summary/Abstract Class switch recombination (CSR) is a genetic process where a B cell switches antibody isotype production through site-specific intra-chromosomal DNA rearrangement stimulated by the formation of DNA double-strand breaks (DSBs) at the immunoglobulin heavy chain (IgH) locus. DSBs are normally repaired by the non-homologous end-joining (NHEJ) and alternative-end joining (alt-EJ) DNA repair pathways. During CSR, DSB formation is highly regulated involving a complex interplay of transcriptional activation, protein recruitment and chromatin reorganization. Understanding the factors regulating DSB formation and repair has a high impact on lymphomagenesis. R loops are three stranded RNA:DNA hybrid structures formed at IgH during CSR. While R loops are implicated in promoting DSB formation at IgH, their role in class switch recombination remains undefined. We find that mice defective for R loop removal are proficient at class switch recombination, however B cells contain unrepaired breaks and chromosome fusions at IgH. Recurrent oncogenic translocations involving IgH distinguish many human lymphoid malignancies. These translocations originate from mis-repaired DNA double stand breaks (DSBs) generated during normal lymphocyte development. Our goal is to determine how persistent R loops impede DNA repair during CSR, and the role R loop metabolism plays in suppressing genome instability at IgH. We hypothesize that persistent R loops block efficient DNA repair by non-homologous end joining at the immunoglobulin heavy chain locus during class switch recombination, leading to persistent, unrepaired breaks. To test this hypothesis, two mouse models will be employed: the SETX mutant lacks the Senataxin (SETX) helicase that unwinds R loops;; and Rnaseh2b is defective for the RNase H2 nuclease that specifically digests the RNA component of R loops (RNH2B). We will functionally dissect the consequences of aberrant R loop formation on DNA repair and chromosome fusions arising during CSR in SETX-/-, RNH2Bf/f, and SETX-/- RNH2Bf/f cells (Aim 1). To define the impact persistent R loops have on NHEJ, we will characterize DNA repair protein recruitment in SETX-/-, RNH2Bf/f, and SETX-/- RNH2Bf/f cells (Aim 2). We will also identify genomic loci involved in IgH translocations using high-throughput genome-wide translocation sequencing (HTGTS-Seq), in collaboration with Dr. Feyredoun Hormozdiari. Finally, we will define the molecular pathways driving the frequent chromosome fusions observed in SETX-/- RNH2Bf/f cells (Aim 3). Our work will define how persistent R loops interfere with class switch recombination, leading to unrepaired breaks, and will uncover the molecular mechanisms promoting chromosome fusions at IgH. Enzymes regulating R loop metabolism will also provide an attractive target for developing novel cancer treatment.