Mutation in the magnesium binding site of hMSH6 disables the hMutSα sliding clamp from translocating along DNA

In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutS alpha, a heterodimer of hMSH2 and hMSH6, In the presence of ATP and magnesium, hMutS alpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutS alpha variant, hMuts alpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with trans location. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The translocation-prone conformation of wild type hMutS alpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gamma S or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutS alpha in the presence and in the absence of magnesium helps explain the functional differences between hMutS alpha variants incapable of binding ATP as compared with those unable to bind the metal ion.