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 hetero dimers, hMutS alpha (hMSH2+hMSH6), which preferentially binds to base-base mismatches and hMutS beta (hMSH2+hMSH3), which binds to insertion/deletion loops. We studied these mismatch binding activities in several human cell l ines with a gel-shift assay using various mismatch oligonucleotides as subs trates, Both hMutS alpha and hMutS beta activities could be detected in var ious human cell lines, In cells with amplified copies of the hMSH3 gene, a large increase in hMutS beta and a reduction in hMutS alpha were observed. To identify the composition of each mismatch binding complex, the protein-D IVA complexes were transferred from gel-shift polyacrylamide get to a polyv inylidene difluoride membrane and were subjected to immunoblot analysis wit h an enhanced chemiluminescence protein detection system, The results clear ly demonstrated that hMutS alpha detected by the gel-shift assay was compos ed of hMSH2 and hMSH6, while hMutS beta was composed of hMSH2 and hMSH3, Ou r data, therefore, support a model whereby formation of hMutS alpha and hMu tS beta is mutually regulated, Combination of a gel-shift assay with immuno blotting (shift-Western assay) proved to be a highly sensitive technique an d should be useful for studying the interactions between DNA and binding pr oteins, including DNA mismatch recognition. (C) 1999 Federation of European Biochemical Societies.