Measurement of DNA mismatch repair activity in live cells.
Loss of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Currently, assays for DNA MMR activity involve the use of cell extracts and are technically challenging and costly. Here, we report a rapid, less labor-intensive method that can quantitatively measure MMR activity in live cells. A G-G or T-G mismatch was introduced into the ATG start codon of the enhanced green fluorescent protein (EGFP) gene. Repair of the G-G or T-G mismatch to G-C or T-A, respectively, in the heteroduplex plasmid generates a functional EGFP gene expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were transfected in parallel into the same cell line and the number of green cells counted by flow cytometry. Relative EGFP expression was calculated as the total fluorescence intensity of cells transfected with the heteroduplex construct divided by that of cells transfected with the homoduplex construct. We have tested several cell lines from both MMR-deficient and MMR-proficient groups using this method, including a colon carcinoma cell line HCT116 with defective hMLH1 gene and a derivative complemented by transient transfection with hMLH1 cDNA. Results show that MMR-proficient cells have significantly higher EGFP expression than MMR-deficient cells, and that transient expression of hMLH1 alone can elevate MMR activity in HCT116 cells. This method is potentially useful in comparing and monitoring MMR activity in live cells under various growth conditions.
Lei
Xiufen
X
Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
Zhu
Yong
Y
Tomkinson
Alan
A
Sun
LuZhe
L
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch; D002352:Carrier Proteins; D045744:Cell Line, Tumor; D018387:Codon, Initiator; D004260:DNA Repair; D005434:Flow Cytometry; D005816:Genetic Complementation Test; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D020180:Heteroduplex Analysis; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018387:Codon, Initiator; D008164:Luminescent Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; D049452:Green Fluorescent Proteins; D000070957:MutL Protein Homolog 1