Abstract 2167 Selecting Mre11 Mutations Through Kinetic Analysis for Use in FRET Assays

As part of the DNA damage repair system, the Mre11/Rad50 (MR) complex is a first-responder to double-stranded DNA breaks. This is important because dsDNA breaks are one of the most serious forms of DNA damage. Rad50 is an ABC ATPase that energizes the nuclease activity of Mre11. The ATPase and nuclease activity have already been thoroughly studied using both ensemble and single molecule assays. Although we know structures that indicate the conformations of the MR complex, the frequency of its conformational changes are still unclear. We plan to study the conformational changes using single-molecule FRET assays to understand the movements of the Rad50 coiled-coil domain, and the movement of Mre11 with respect to Rad50, in parallel to the complex's ATPase and nuclease activity. To study these changes, we selected places in both proteins to mutate and label for further analysis in FRET assays. However, thus far we have mainly focused on Mre11 mutations. To get the best results in FRET assays, we chose positions based on distance in accordance with the predicted structures and movement while taking into consideration that FRET works best within 10 nm. Then we tested these mutants to determine how their ATPase and nuclease activity compare with that of the wild type. We have just gotten to the point of labeling each site using click chemistry and an unnatural amino acid in Mre11 and hope to soon run cuvette FRET assays to find the best mutants for single molecule FRET.