Structural Biology Studies of a Large DNA Repair Complex

Our research program revolves around understanding the relationship between structure, dynamics, and function. We are particularly interested in understanding this relationship in large macromolecular assemblies, which have been recalcitrant to detailed structure and dynamics studies in the past. Our strategy is to couple high-resolution methyl-based nuclear magnetic resonance (NMR) spectroscopy, which is capable of probing proteins and their complexes up to ~1 MDa in size, with biochemical and biophysical techniques to better understand function. Our current focus is the universally conserved and essential DNA double strand break (DSB) repair complex Mre11-Rad50-Nbs1 (MRN). This protein complex is at the heart of detecting DNA DSBs and initiating the process of their repair. Several disease mutations have been noted in MRN, which give rise to immunodeficiency, developmental and neurodegenerative disorders, and a predisposition to certain cancers. Other sporadic mutations in Mre11 and Rad50 have been found in a number of different cancers. Bacterial and archaeal model systems, which lack Nbs1, have been the focus of the existing body of X-ray crystallography and biochemical studies that suggest a role for protein motions in choreographing the various functions of the Mre11- Rad50 (MR) core complex. Yet, many questions still remain about the interplay of protein structures and motions and how these relate to and control MR activity. Over the next five years, our goal is to determine solution state models of key MR assemblies complete with substrate DNAs that mimic different types of DNA DSBs and to characterize the protein dynamics that occur within these complexes. The NMR-based studies will be complemented with a variety of in vitro biophysical and biochemical techniques to further probe domain motions and the wide array of MR activities, as well as in vivo studies in yeast to place these motions and activities into the context of overall DNA DSB repair. We will also extend these studies to include disease mutations within MR, which will not only allow us to understand how these alterations corrupt MR function and lead to disease but will also provide additional avenues for probing the structures, dynamics, and functional relationships within this complex. Our long-term goals seek to move beyond the core MR complex. Although the MR studies proposed herein will be performed on the simplified construct of Rad50, which has been used in all previous x-ray crystallographic studies of MR, we aim to perform, for the first time, similar studies using full-length Rad50. In total, our research program aims to better understand how macromolecular assemblies use protein motions to regulate their functions, and in the process of applying our program to MRN, we will determine the effect that large and small scale motions have in controlling the first steps in MRN-mediated DNA DSB repair.