Identification of the protein components of mismatch binding complexes in human cells using a gel-shift assay

In eukaryotes, mismatch recognition is thought to be mediated by two heterodimers, hMutSalpha (hMSH2+hMSH6), which preferentially binds to base-base mismatches and hMutSbeta (hMSH2+hMSH3), which binds to insertion/deletion loops. We studied these mismatch binding activities in several human cell lines with a gel-shift assay using various mismatch oligonucleotides as substrates. Both hMutSalpha and hMutSbeta activities could be detected in various human cell lines. In cells with amplified copies of the hMSH3 gene, a large increase in hMutSbeta and a reduction in hMutSalpha were observed. To identify the composition of each mismatch binding complex, the protein-DNA complexes were transferred from gel-shift polyacrylamide gel to a polyvinylidene difluoride membrane and were subjected to immunoblot analysis with an enhanced chemiluminescence protein detection system. The results clearly demonstrated that hMutSalpha detected by the gel-shift assay was composed of hMSH2 and hMSH6, while hMutSbeta was composed of hMSH2 and hMSH3. Our data, therefore, support a model whereby formation of hMutSalpha and hMutSbeta is mutually regulated. Combination of a gel-shift assay with immunoblotting (shift-Western assay) proved to be a highly sensitive technique and should be useful for studying the interactions between DNA and binding proteins, including DNA mismatch recognition.