Conformational heterogeneity in the MR complex underlie its functions in DNA double-strand break repair

Friday, October 7, 2022 - 3:20pm
Event Type: 

Dr. Michael P. Latham, Department of Biochemistry, Molecular Biology, and Biophysics, the University of Minnesota

Host: Dr. Venditti

Our genomic DNA is under constant assault from internal and external stressors. The failure to repair damage to our DNA can result in harmless mutations or more serious alterations that are the driving force for the progression of cancer. The most deleterious DNA lesion is the double strand break, where the phosphodiester bond on both strands of the DNA is cleaved, because a direct template for repair is not directly available. The Mre11-Rad50 (MR) complex is an essential and universally conserved protein complex that is among the first-responders to the site of DNA double strand breaks. X-ray crystallography and cryo-electron microscopy have revealed a number of structures of the MR complex that are modulated by ATP and DNA binding to the MR complex. We combine sensitive methyl-based NMR experiments, on highly deuterated, side-chain methyl-group 13CH3-labeled samples of the 120 kDa MR complex, with other biophysical and biochemical techniques to understand how these various structures and dynamics affect MR cooperativity, allosteric regulation, and function. First, I will discuss recent models we have determined of dsDNA bound to the ATP-free form of the MR complex. Using these models, we have generated a number of mutants that have illuminated the relationship between Rad50 ATP hydrolysis and Mre11 nuclease activity. Second, I will describe a new two-site protein ligation strategy that we have developed which is enabling studies into long-range allostery within the complex. In total, this work demonstrates the role of conformational heterogeneity for the functions of the MR complex in DNA double-strand break repair.