Principal Investigator: Wu, Xiaohua Project Summary Gross chromosomal rearrangement (GCR) is a hallmark of cancer cells and the cause of structural variant formation in genetic disorders. DNA double-strand breaks (DSBs) are a major source to induce GCR, but the details on how the DSB repair process leads to genome instability in cancers and other genetic disorders remains elusive. Microhomologies are prevalent at chromosomal break junctions in cancer and other human diseases, suggesting that microhomology-mediated end joining (MMEJ) is one major player to mediate GCRs. Repetitive sequences are abundant, constituting about half of the human genome, which raises the question of how recombination between repetitive sequences is suppressed to prevent genome instability. In this proposal, we aim to investigate the mechanisms underlying MMEJ and repeat-mediated recombination in mammalian cells. We have established multiple EGFP-based DSB repair reporter systems, which enable us to study different DSB repair pathways in mammalian cells. By using newly established MMEJ reporters, we identified new players in the MMEJ pathway. We will investigate how these MMEJ players regulate MMEJ to balance its usage and its mutagenic consequences. We will also study the biological role of MMEJ in repairing replication- associated DSBs and in coping with replication and oncogenic stress, and address how MMEJ contributes to genome instability and the oncogenic process. Additionally, we will determine the mechanisms of repeat-mediated recombination in causing deletions and translocations, and study the role of heteroduplex rejection in preventing recombination among divergent repeats. These studies will generate much-needed new knowledge to fill the gap in our understanding of DSB repair mechanisms and the cause of genome instability that is associated with the oncogenic process and other genetic disorders.