Abstract
Analyses in vitro of correction of DNA mismatches have been pivotal in biochemical dissection of mismatch repair pathways. However, the complex procedures needed to prepare DNA substrates for mismatch repair have posed substantial barriers to investigators who wish to pursue such analyses. Here we describe a simple, efficient way to prepare a variety of mismatched DNA substrates. We use in our procedure high-copy-number pUC19-derived plasmids, and a newly commercially available endonuclease N.BstNBI that makes site-specific single-strand nicks. The ability to prepare large substrate quantities in a relatively short time and to construct wider ranges of different mismatches in various sequence contexts will facilitate future research.
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Potter, D. (1999) Colorectal cancer: molecules and populations. J. Natl. Cancer Inst. 91, 916–932.
Branch, P., Aquilina, G., Bignami, M., and Karran, P. (1993) Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 362, 652–654.
Kat, A., Thilly, W. G., Fang, W.-H., Longley, M.J., Li, G.-M., and Modrich, P. (1993) An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair. Proc. Natl. Acad. Sci. USA 90, 6424–6428.
Koi, M., Umar, A., Chauhan, D., Cherian, S. P., Carethers, J. M., Kunkel, T. A. and Boland, C. R. (1994) Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N′-nitro-N-nitrososguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation. Cancer Res. 54, 4308–4312.
Risinger, J. I., Umar, A., Barrett, J. C., and Kunkel, T. A. (1995) A hPMS2 mutant cell line is defective in strand-specific mismatch repair. J. Biol. Chem. 270, 18183–18186.
Aquilina, G., Hess, P., Fiumicino, S., Ceccotti, S. and Bignami, M. (1993) A mutator phenotype characterizes one of two complementation groups in human cells tolerant to methylation damage. Cancer Res. 55, 2569–2575.
Hampson, R., Humbert, O., MacPherson, P., Aquilina, G., and Karran, P. (1997) Mismatch repair defects and 0 6-methylguanine-DNA methyltransferase expression in acquired resistance to methylating agents in human cells. J. Biol. Chem. 272, 28596–28606.
Umar, A., Koi, M., Risinger, J. I., Glaab, W. E., Tindall, K. R., Kolodner, R. D., et al. (1997) Correction of hypermutability, MNNG resistance and defective DNA mismatch repair by introducing chromosome 2 into tumor cells with mutations in MSH2 and MSH6. Cancer Res. 57, 3949–3955.
Corrette-Bennett, S. E. and Lahue, R. S. (1999) Mismatch repair assay. In DNA Repair Protocols: Eukaryotic Systems (Henderson, D.S., ed), Totowa, NJ; Humana Press, pp 121–132.
Holmes, J., Clark, S., and Modrich, P. (1990) Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc. Natl. Acad. Sci. USA 87, 5837–5841.
Modrich, P. (1997) Strand-specific mismatch repair in mammalian cells. J. Biol. Chem. 272, 24727–24730.
Lanue, R. S. and Modrich, F. (1989) DNA mismatch correction in a defined system. Science 245, 160–164
Parsons, R., Li, G-M., Longley, M. J., Fang, W-H., Rapadopoulos, N., Jen, J., et al. (1993) Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell 75, 1227–1236
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Supported in part by NIH grant ESO94848 to J.B.H. This is Technical Report No. 11680 from the Oregon Agricultural Experiment Station
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Wang, H., Hays, J.B. Preparation of DNA substrates for in vitro mismatch repair. Mol Biotechnol 15, 97–104 (2000). https://doi.org/10.1385/MB:15:2:97
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DOI: https://doi.org/10.1385/MB:15:2:97