Effect of nucleotide binding on Rad50 conformational state, multimeric state, and DNA binding ability
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The ability of an organism to swiftly repair double-stranded breaks (DSBs) in DNA is a crucial process that, if absent, can result in genomic instability. The Mre11/Rad50 protein complex is highly conserved and plays a key role in sensing, processing, and repairing DNA DSBs. Rad50 is known to be necessary for the DNA end-bridging catalyzed by the MRN complex, and association of the broken DNA ends is essential to prevent loss of chromosome fragments in vivo. The Rad50 protein contains a large coiled-coil domain and ATP-binding motifs, with an overall structure similar to the Structural Maintenance of Chromosomes (SMC) family of proteins. Rad50 undergoes ATP-dependent homodimerization, which creates a potential DNA binding cleft. Recently, Rad50 has been shown to have adenylate kinase activity in addition to the previously known ATPase activity. It is still unknown what role ATP-induced dimerization and conformational changes play in pfRad50’s various activities. We hypothesize that dimerization is needed for DNA binding, and that Rad50 requires large conformational changes for proper function in its part in pfMR exonuclease activity. Here, we use site-directed mutagenesis to create Rad50 mutants that have cysteines placed in structurally relevant portions of the protein. With these cysteines, we used disulfide crosslinking, fluorescence, and Fluorescence Resonance Energy Transfer (FRET) to detect if changes in conformation or multimeric state of Rad50 are necessary for adenylate kinase activity, ATPase activity, and DNA binding.