The research goal of this project is to achieve a detailed understanding of how the Mre11/Rad50 (MR) DNA repair complex carries out and regulates its enzymatic activity. The MR complex is involved in the processing of DNA double-strand breaks (DSBs), which are among the most harmful forms of DNA damage and their improper repair may lead to cellular dysfunction or death. The completion of this project is expected to provide insights into how organisms maintain their genetic material over many generations in the face of internal and external DNA damaging agents. This project will also provide high quality training experiences for high school, undergraduate, and graduate students. The PI actively participates in several internship programs with the goal of increasing the diversity of talented individuals choosing science as a career. The PI is partnering with the Science Bound program at Iowa State University and will create a laboratory module for visiting high school students that involves UV-induced DNA damage and repair. Finally, the PI is working to increase scientific literacy by developing and teaching introductory science lecture and laboratory courses designed for non-science majors.
The overall objective of this research project is to define the catalytic and regulatory mechanisms of the MR complex. Previous studies carried out in the PI's lab strongly suggest that the underlying mechanisms that operate in the MR complex are well conserved among homologs. It was found that the nuclease activity of the bacteriophage T4 MR complex can be altered depending on the nature of the divalent cation used. While Mn2+ supports a robust 3' to 5' dsDNA exonuclease activity, Mg2+ may support a more physiologically relevant nuclease activity. A series of in vivo and in vitro experiments will be carried out with the goal of determining the identity of the divalent cation used by Mre11 to perform its activity in vivo. The PI has proposed that the Rad50 coiled-coil domain alternates between ring-like and parallel conformational states, which are driven by the ATP hydrolysis cycle and serve to regulate the ATPase and nuclease activities of the complex. A combination of biophysical techniques that report on the conformation and dynamics of the coiled-coil domain will be employed to test this hypothesis. The entire ensemble of conformational states that the coiled-coil adopts and their rates of interconversion will be determined. Similar methods will be used to monitor conformational changes in Mre11 and to determine how these are controlled by the activity of Rad50. The results of these studies are expected to significantly enhance our understanding of the MR complex and how its activities are coordinated during DSB repair.
