LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex

Abstract

The Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional ‘partially open’ conformation using Luminescence Resonance Energy Transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with SAXS data which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.

Dynamic Properties of the DNA Damage Response Mre11/Rad50 Complex

“…Performing luminescence resonance energy transfer (LRET) experiments allowed us to obtain distance restraints that drove the computational reconstruction of three different states of MRad50 complexes in a dynamic equilibrium that is independent from the nature of the nucleotide and is observed in the presence of DNA as well [27,47]. It is unclear if the so-called 'partially open' state is only a transition state or if it is competent for specific functions exerted by the complex.…”