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.
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
E
E
Nakajima
M
M
Ikejima
A
A
Watanabe
T
T
Shimada
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