Project Summary/Abstract DNA alkylation damage comprises a class of prevalent, harmful nucleobase modifications that occur thousands of times per cell per day in the human body as a result of endogenous and exogenous sources. Left unrepaired, DNA alkylation damage can result in genetic mutations, the inhibition of DNA replication, and cell death. Several DNA repair pathways have evolved to reverse the numerous DNA modifications that result from alkylation damage. While the repair enzymes in these pathways are well studied, much less is known about the upstream signaling events that initiate DNA repair and localize repair complexes to damage sites. It was recently shown that the ALKBH3-ASCC DNA repair complex is recruited to alkylation damage sites by binding chains of the protein ubiquitin that are assembled in proximity to the DNA lesions. The protein ASCC2 is responsible for binding the polyubiquitin chains that localize the ALKBH3-ASCC complex. A vast array of different types of polyubiquitin chains are present in cells, however, and it is unclear how ASCC2 selectively recognizes the K63- linked polyubiquitin chains that signal alkylation damage. The PI proposes to use a combination of structural biology, cell biology, and biophysics to investigate ASCC2’s selectivity for K63-linked polyubiquitin chains and the dependence of ALKBH3-ASCC complex localization on the unique ubiquitin-binding properties of ASCC2. The specific aims of the project are: 1) to identify the novel ASCC2:ubiquitin binding interface that imparts enhanced affinity for polyubiquitin chains, 2) to determine the structural basis of ASCC2’s specificity for binding K63-linked polyubiquitin chains, and 3) to quantify the contribution of ASCC2’s ubiquitin-binding properties to DNA alkylation damage repair. Investigating the outstanding questions associated with DNA alkylation damage repair will allow clinicians to better understand diseases that result from defects in alkylation damage repair pathways and to more effectively deploy alkylating agents as therapeutics, especially for the treatment of cancer. Furthermore, these experiments will also provide valuable research opportunities for students at Mount St. Mary’s University (MSMU), where substantial populations of the biology, chemistry, and biochemistry majors are first-generation college students (16.7%), students of color (42.5%), or students from moderate- or low-income families (27.1 % Pell Grant recipients). Overall, the proposed experiments will address a lack of knowledge in the current understanding of DNA alkylation damage repair while greatly enhancing research opportunities for students at MSMU.