Abstract: Msh4-Msh5 or MutSy is an important protein heterodimer that belongs to the family of MutS repair proteins that are involved in post-replicative mismatch repair and help to maintain genome integrity and cellular stability. Msh4-Msh5 is critical for stabilizing joint molecule DNA recombination intermediates, for promoting crossover recombination, and for proper assembly of the synaptonemal complex. Previous experiments with the MutS and Msh2-Msh6 proteins, have shown that the subunits participate asymmetrically in ATP binding and hydrolysis as well as DNA binding. We are therefore seeking to elucidate whether a similar functional asymmetry in Msh4-Msh5 by generating mutants via site-directed mutagenesis. Key residues in ATP binding and hydrolysis will are mutated to disrupt the activity of one subunit, while the other subunit remains unchanged. After introducing the mutations in the Walker A (ATP binding) and Walker B (ATP hydrolysis) motif of each subunit, ATP assays can be utilized to characterize ATP binding and hydrolysis for each individual subunit. Optimization of protein solubility and protein expression were tangentially studied for protein purification of Msh4-Msh5 by using the Maltose Binding Protein affinity tag, which increases protein solubility, attached at the N-terminus of Msh4.
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