Regulation of mismatch repair protein MutSα functions by its Walker A and Walker B motifs

Abstract

The mismatch recognition protein MutSα is an MSH2‐MSH6 heterodimer. Mismatch binding by MutSα triggers a series of downstream mismatch repair (MMR) reactions, including interacting and communicating with other MMR proteins by sliding along the DNA helixes. These MutSα biochemical functions rely on its ATP/ADP binding and ATPase activities. Like many ATPases, both MSH2 and MSH6 subunits contain a Walker A motif and a Walker B motif. However, how these Walker motifs contribute to the MutSα functions is not fully understood. To address this issue, a series of MutSα mutants that contain a lysine (K) to alanine substitution in Walker A motif and a glutamate (E) to K substitution in Walker B motif of either MSH2 or MSH6, or both were analyzed for their MMR activities. We show here that MSH2 mutations in Walker A or B motif block MutSα ATP hydrolysis, suggesting that the MSH2 Walker motifs regulate MutSα ATPase activity. Interestingly, the E‐K substitutions in the Walker B motif produce a mutant MutSα that, instead of sliding off, undergoes direct dissociation from a mismatch‐containing DNA molecule regardless of the presence of magnesium. This indicates that the E residue in the Walker B motif of either MSH2 or MSH6 is required for magnesium binding, and is indispensable for MutSα conformational change to form a sliding clamp. Our work provides new insights into how the Walker motifs contribute to MutSα activities in MMR.