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.
Nakajima
E
E
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Ikejima
M
M
Watanabe
A
A
Shimada
T
T
D020137:Base Pair Mismatch; D002474:Cell-Free System; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004351:Drug Resistance; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D009687:Nuclear Proteins / Q000302:isolation & purification; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D008727:Methotrexate