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.

[1]  M. Capecchi,et al.  Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei , 1985, Molecular and cellular biology.

[2]  J. H. Taylor,et al.  One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Jiricny,et al.  A specific mismatch repair event protects mammalian cells from loss of 5-methylcytosine , 1987, Cell.

[4]  K. M. Mulder,et al.  Effects of growth stimulatory factors on mitogenicity and c-myc expression in poorly differentiated and well differentiated human colon carcinoma cells. , 1989, Molecular endocrinology.

[5]  P. Modrich,et al.  Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[6]  T. Kunkel,et al.  Heteroduplex repair in extracts of human HeLa cells. , 1991, The Journal of biological chemistry.

[7]  A. Tomkinson,et al.  DNA ligase III is the major high molecular weight DNA joining activity in SV40-transformed human fibroblasts: normal levels of DNA ligase III activity in Bloom syndrome cells. , 1993, Nucleic acids research.

[8]  Bert Vogelstein,et al.  Hypermutability and mismatch repair deficiency in RER+ tumor cells , 1993, Cell.

[9]  D. Ward,et al.  Mutation in the DNA mismatch repair gene homologue hMLH 1 is associated with hereditary non-polyposis colon cancer , 1994, Nature.

[10]  T. Kunkel,et al.  Defective mismatch repair in extracts of colorectal and endometrial cancer cell lines exhibiting microsatellite instability. , 1994, The Journal of biological chemistry.

[11]  Sajeev P. Cherian,et al.  Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation. , 1994, Cancer research.

[12]  D. S. Hsu,et al.  Substrate spectrum of human excinuclease: repair of abasic sites, methylated bases, mismatches, and bulky adducts. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Fleischmann,et al.  Mutation of a mutL homolog in hereditary colon cancer. , 1994, Science.

[14]  J D Tucker,et al.  Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells. , 1995, Nucleic acids research.

[15]  J. Jiricny,et al.  GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells. , 1995, Science.

[16]  P. Modrich,et al.  Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[17]  P. Modrich,et al.  Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells. , 1995, Science.

[18]  P. Karran,et al.  DNA mismatch binding defects, DNA damage tolerance, and mutator phenotypes in human colorectal carcinoma cell lines. , 1995, Cancer research.

[19]  Asad Umar,et al.  Meiotic Pachytene Arrest in MLH1-Deficient Mice , 1996, Cell.

[20]  T. Kunkel,et al.  DNA-replication fidelity, mismatch repair and genome instability in cancer cells. , 1996, European journal of biochemistry.

[21]  J. Jiricny,et al.  hMutSβ, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA , 1996, Current Biology.

[22]  P. Modrich,et al.  Mismatch repair in replication fidelity, genetic recombination, and cancer biology. , 1996, Annual review of biochemistry.

[23]  J. Eshleman,et al.  Mismatch repair defects in human carcinogenesis. , 1996, Human molecular genetics.

[24]  P. Macpherson,et al.  hMSH2-independent DNA Mismatch Recognition by Human Proteins (*) , 1996, The Journal of Biological Chemistry.

[25]  J. Winkles,et al.  Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression. , 1996, The Biochemical journal.

[26]  G. Tocco,et al.  DNA Replication and Postreplication Mismatch Repair in Cell-Free Extracts from Cultured Human Neuroblastoma and Fibroblast Cells , 1997, The Journal of Neuroscience.

[27]  P. Modrich.  Strand-specific Mismatch Repair in Mammalian Cells* , 1997, The Journal of Biological Chemistry.

[28]  M. Meyers,et al.  Cell cycle regulation of the human DNA mismatch repair genes hMSH2, hMLH1, and hPMS2. , 1997, Cancer research.

[29]  Peter Beighton,et al.  de la Chapelle, A. , 1997 .

[30]  M. Loda,et al.  Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. , 1997, Cancer research.

[31]  C. Fisher,et al.  Modification of a PCR-based site-directed mutagenesis method. , 1997, BioTechniques.

[32]  S N Thibodeau,et al.  Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. , 1998, Cancer research.

[33]  J. Jiricny,et al.  Mismatch repair deficiency associated with overexpression of the MSH3 gene. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Arends,et al.  DNA mismatch repair and colorectal cancer , 1998, The Journal of pathology.

[35]  R. Fishel.  Mismatch repair, molecular switches, and signal transduction. , 1998, Genes & development.

[36]  T. Prolla,et al.  DNA mismatch repair and cancer. , 1998, Current opinion in cell biology.

[37]  J. Herman,et al.  Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Nickoloff,et al.  Efficient repair of all types of single-base mismatches in recombination intermediates in Chinese hamster ovary cells. Competition between long-patch and G-T glycosylase-mediated repair of G-T mismatches. , 1998, Genetics.

[39]  J. G. Park,et al.  Growth suppression mediated by transfection of wild-type hMLH1 in human cancer cells expressing endogenous truncated hMLH1 protein. , 1998, International journal of oncology.

[40]  L. Kasturi,et al.  Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Tytell,et al.  Germ-line msh6 mutations in colorectal cancer families. , 1999, Cancer research.

[42]  A. Thorburn,et al.  Apoptosis induced by overexpression of hMSH2 or hMLH1. , 1999, Cancer research.

[43]  Josef Jiricny,et al.  Identification of hMutLβ, a Heterodimer of hMLH1 and hPMS1* , 1999, The Journal of Biological Chemistry.

[44]  R. Liskay,et al.  The human MLH1 cDNA complements DNA mismatch repair defects in Mlh1-deficient mouse embryonic fibroblasts. , 1999, Cancer research.

[45]  H. Hollema,et al.  Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations. , 1999, American journal of human genetics.

[46]  L. Thompson,et al.  XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. , 1999, Genes & development.

[47]  G. Marsischky,et al.  Eukaryotic DNA mismatch repair. , 1999, Current opinion in genetics & development.

[48]  A. Sepulveda,et al.  Identification of a Second MutL DNA Mismatch Repair Complex (hPMS1 and hMLH1) in Human Epithelial Cells* , 2000, The Journal of Biological Chemistry.

[49]  Andreas D. Baxevanis,et al.  MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability , 2000, Nature Genetics.

[50]  P. Frit,et al.  Scientific Report , 2022 .

[51]  J. Willson,et al.  Heterogeneity of human colon carcinoma , 2004, Cancer and Metastasis Reviews.