DNA replication errors that escape the proofreading activity of the replicative DNA polymerase are repaired by DNA mismatch repair (MMR). The initiation of MMR in Escherichia coli involves the recognition of the mismatch by MutS, binding of MutL, and activation of the endonuclease MutH which incises DNA at a hemi-methylated GATC site. MutS exists in a dimer-tetramer equilibrium, but the function of the tetramer during MMR remains unknown. Here, we used in vitro MutH activation assays to examine the role of MutS in the reaction steps that couple mismatch recognition to daughter strand incision. To study the behavior of different MutS oligomers, we used obligate dimers and tetramers and quantified GATC site incision on circular and linear DNA substrates. Especially in the presence of free DNA ends, MutS tetramers mediate more efficient incision than MutS dimers, likely due to tetramers diffusing slower and therefore being more successful in assembling the active incision complex before dissociating from the DNA at the ends. Likewise, we observed that MutS tetramers have a higher preference for nicking the GATC site close to the mismatch than dimeric MutS. Through probabilistic modeling, we show that this increased preference is consistent with a fourfold decrease in diffusion constant for the tetramer compared to the dimer. We propose that during mismatch repair, DNA excision tracts resulting from MutS tetramer-mediated incision will be shorter than those mediated by the dimer, and that this explains the reported higher repair efficiencies of wild-type MutS compared to the dimer.