Published online 12 July 2004
Nucleic Acids Research, Vol. 32 No. 12 © Oxford University Press 2004; all rights reserved
Measurement of DNA mismatch repair activity in live cells
Department of Cellular and Structural Biology and 1 Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA and 2 Department of Pharmacology, University of Kentucky College of Medicine, Lexington, KY 40536-0036, USA
* To whom correspondence should be addressed. Tel: +1 210 567 5746; Fax: +1 210 567 3803; Email: sunl{at}uthscsa.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received April 4, 2004; Revised June 7, 2004; Accepted June 18, 2004
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.
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