Project Summary/Abstract Over the past 20 years, the protein ASCC2 has been shown to contribute to the localization of numerous multiprotein complexes involved in pathways such as DNA alkylation damage repair and the stalled ribosome response. A common feature of several of these pathways is that they assemble K63-linked polyubiquitin chains which are recognized by ASCC2 to recruit the appropriate multiprotein complexes. ASCC2 utilizes a CUE domain to bind the K63-linked polyubiquitin chains, however, CUE domains are typically promiscuous ubiquitin binders, and it is unclear how ASCC2 specifically recognizes K63-linked polyubiquitin chains among the myriad types of polyubiquitin chains present in the cell. The overarching goal of the proposed experiments is to elucidate the structural basis for ASCC2’s specificity for binding K63-linked polyubiquitin chains with the following specific aims: 1) identify the intermolecular interactions that mediate binding between ASCC2 and K63-linked polyubiquitin chains using nuclear magnetic resonance (NMR) spectroscopy, 2) use integrative modeling to create a representation of the interaction between ASCC2 and K63-linked polyubiquitin chains that agrees with our experimentally determined NMR, mutagenesis, and small angle X-ray scattering data, and 3) quantify the functional significance of the predicted interactions between ASCC2 and K63-linked polyubiquitin chains using in vitro and cell-based assays. These studies are part of a broader effort to better understand the biological pathways that depend on ASCC2’s ubiquitin-binding abilities and to diagnose diseases associated with the malfunction of these pathways. For example, in the DNA alkylation damage repair pathway, mutations that inhibit the formation of the K63-linked polyubiquitin chains that recruit ASCC2, and mutations that inhibit the interaction between ASCC2 and other members of the ALKBH3-ASCC DNA repair complex, have recently been associated with genetic diseases due to research in this area. Additionally, the proposed studies will provide valuable research opportunities for students at Mount St. Mary’s University (MSMU). Collaborations with scientists at Johns Hopkins University, Washington University in St. Louis, and the SIBYLS beamline at the Lawrence Berkeley National Laboratory will allow the MSMU undergraduate researchers access to scientific instrumentation that is not available on their home campus. Overall, the proposed studies will further our understanding of how ASCC2 targets multiprotein complexes to sites marked by K63-linked polyubiquitin chains while greatly enhancing the research opportunities available for students at MSMU.