Our research program aims to understand the relationship between macromolecular structure, dynamics, and function, with a particular focus on large nucleic acid binding proteins. To this end, we use sensitive methyl-based solution-state nuclear magnetic resonance (NMR) spectroscopy, which is capable of probing the structure and dynamics of proteins and their complexes with masses >1 MDa. We couple NMR spectroscopy with other powerful biophysical techniques to probe structure and dynamics and then employ biochemical and in vivo activity assays to better understand function. Our strategy relies on rationally designing mutations to disrupt specific interactions within the protein/protein complex as well as naturally occurring mutations found in disease states to ‘break’ the structure/dynamics/function relationship in order to piece together how the protein complex works. Currently, our primary focus is the MRE11-RAD50- NBS1/Xrs2 (MRN/X) protein complex, which plays a central role in the DNA double strand break (DSB) repair response. The MRN/X complex is essential for detecting and repairing DNA DSB breaks, as well as for signaling their presence to the rest of the DNA DSB response. Additionally, the MRN/X complex has roles in DNA replication and telomere maintenance. Not surprisingly then, mutations in MRN/X have been found in many types of cancer and in diseases characterized by immunodeficiency, neuronal and cerebral degeneration, a sensitivity to ionizing radiation, and oncogenesis. To fully understand how the MRN/X complex performs all of its functions, it is necessary to determine structural models in the absence and presence of its various substrates. Indeed, structural biology techniques, including NMR spectroscopy, have been used to study the MRN/X complex and have revealed a diverse set of structures. Though insightful, these models have raised more questions about how the structures are specifically involved in MRN/X function, the relative populations of these heterogeneous structures in solution, the kinetics for the interconversion between these structures, and short and long-range allosteric communication within them. In the next five years, our goal is to bring clarity to each of these questions by applying our comprehensive biophysical and biochemical research strategy. In general, we strive to better understand how protein complexes use a diverse set of structures and biochemical activities to perform and coordinate complex biological functions. These proposed studies will provide an unparalleled view into MRN/X function in DNA DSB repair.