Fig. 3: Sliding-deficient MutSα is defective in MMR.
From: Mispair-bound human MutS–MutL complex triggers DNA incisions and activates mismatch repair
a Amino acid sequences and positions of the Walker A motif in the MSH2 and MSH6 subunits of MutSα. Mutagenesis was focused on methionine (M) and glycine (G) (in blue) in the WT sequence, with the corresponding mutated residues in red. b Structures of Walker A motif of MSH6. Nucleotide-free (upper panel) and ADP-bound (middle panel) MSH6 structures (PDB: 2O8E and 2O8B) are shown in the blue cartoon. The conserved residues and ADP are depicted as sticks and balls and labeled. MG to DA mutations (highlighted in cyan, bottom panel) stabilize the helical conformation, thus making the protein resistant to nucleotide binding. c ATPase activities of WT and mutant MutSα proteins. d ATP-binding activity of WT and mutant MutSα proteins with or without mismatched DNA. Proteins were incubated with [γ-32P]ATP, followed by UV cross-linking and SDS-PAGE. e EMSA analysis to determine ATP-dependent dissociation of WT and mutant MutSα proteins from a 36-bp heteroduplex DNA. f In vitro MMR assay to determine the ability of individual MutSα proteins to restore MMR in MutSα-deficient nuclear extract.
