The genetic lesions that promote tumorigenesis, including chromosomal rearrangements, are generated primarily by illegitimate DNA repair. In this renewal application, we will continue to investigate the pathological consequences of aberrant DNA double-strand break (DSB) repair. This highly collaborative and integrated program will elucidate how DNA sequence, chromatin accessibility and nuclear organization influence the fate and pathological outcomes of DSBs in a cell type and cell cycle dependent manner. We will continue to use a combination of genetics in yeast and mouse models, biochemistry and cell biology. The application of next generation technologies together with high-throughput genomics approaches provides an unprecedented wealth of information about genomic instability in cancer genomes. We propose to leverage our multifaceted experimental approach with the strong genomics and computational biology components that pervade the Program. Drs. Rabadan and Baer will study mutation signatures - the statistically enriched patterns of DNA substitutions and rearrangements common across tumors - associated with specific BRCA1 deficiencies. They will also characterize mutation signatures generated by the other three Projects. Collectively these data will help deconvolute the complex mutational landscape of human tumors. Drs. Symington and Ciccia will address the nature of the initiating DNA lesions resulting in chromosome rearrangements, focusing on DNA replication errors in repair-deficient yeast and genome-edited mammalian cells. Dr. Sha will connect chromatin accessibility, locally and globally, with its propensity to break and yield translocations; pathological translocations at the immunoglobulin and T cell receptor loci during lymphocyte maturation fuel lymphoma development. Specifically, Dr. Zha will elucidate how ATM deficiencies favor translocations in a cell type and cell cycle dependent manner. Finally, Drs. Gautier and Gottesman will study how the spatial organization of the nucleus modulates DNA repair. Specifically, they will determine how nuclear actin and WASP, the gene mutated in Wiskott-Aldrich Syndrome, enables resection and influences chromosome translocations following DSBs or genotoxic lesions triggered by topoisomerase inhibition. The four Projects will be supported by two scientific Cores. All leaders and co-leaders have a strong history and record of productive collaboration.