Abstract
DNA methyltransferases are important enzymes and their inhibition has many potential applications. The investigation of DNA methyltransferases as well as screening for potential inhibitors requires specialized enzyme assays. In this chapter, we describe three DNA methyltransferase assays, each of them based on a different method: (1) An assay using radioactively labeled AdoMet and biotinylated DNA substrates that is ideal for enzymatic characterization of these enzymes. (2) An assay using bisulfite conversion of in vitro methylated DNA that is ideal to determine details of the methylation pattern introduced by DNA-(cytosine C5)-methyltransferases. (3) A novel fluorescence-coupled, restriction-based assay suitable for high-throughput screening of DNA methyltransferase inhibitors.
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References
Miranda, T. B., and Jones, P. A. (2007) DNA methylation: the nuts and bolts of repression, J Cell Physiol 213, 384–390.
Jeltsch, A. (2002) Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases, Chembiochem 3, 274–293.
Hermann, A., Gowher, H., and Jeltsch, A. (2004) Biochemistry and biology of mammalian DNA methyltransferases, Cell Mol Life Sci 61, 2571–2587.
Jeltsch, A., Jurkowska, R. Z., Jurkowski, T. P., Liebert, K., Rathert, P., and Schlickenrieder, M. (2007) Application of DNA methyltransferases in targeted DNA methylation, Appl Microbiol Biotechnol 75, 1233–1240.
Lobner-Olesen, A., Skovgaard, O., and Marinus, M. G. (2005) Dam methylation: coordinating cellular processes, Curr Opin Microbiol 8, 154–160.
Jeltsch, A., and Jurkowska, R. Z. (2010) DNA Methylation, in The Chemical Biology of Nucleic Acids (Mayer, G., Ed.), pp 307–322, Wiley-VCH.
Robertson, K. D. (2005) DNA methylation and human disease, Nat Rev Genet 6, 597–610.
Jones, P. A., and Baylin, S. B. (2007) The epigenomics of cancer, Cell 128, 683–692.
Feinberg, A. P., and Tycko, B. (2004) The history of cancer epigenetics, Nat Rev Cancer 4, 143–153.
Sharma, S., Kelly, T. K., and Jones, P. A. (2010) Epigenetics in cancer, Carcinogenesis 31, 27–36.
Cheng, X. (1995) Structure and function of DNA methyltransferases, Annu Rev Biophys Biomol Struct 24, 293–318.
Oakeley, E. J. (1999) DNA methylation analysis: a review of current methodologies, Pharmacol Ther 84, 389–400.
Fraga, M. F., and Esteller, M. (2002) DNA methylation: a profile of methods and applications, Biotechniques 33, 632, 634, 636–649.
Bird, A. P., and Southern, E. M. (1978) Use of restriction enzymes to study eukaryotic DNA methylation: I. The methylation pattern in ribosomal DNA from Xenopus laevis, J Mol Biol 118, 27–47.
Bird, A. P. (1978) Use of restriction enzymes to study eukaryotic DNA methylation: II. The symmetry of methylated sites supports semi-conservative copying of the methylation pattern, J Mol Biol 118, 49–60.
Singer, J., Roberts-Ems, J., and Riggs, A. D. (1979) Methylation of mouse liver DNA studied by means of the restriction enzymes msp I and hpa II, Science 203, 1019–1021.
Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., Molloy, P. L., and Paul, C. L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands, Proc Natl Acad Sci U S A 89, 1827–1831.
Clark, S. J., Harrison, J., Paul, C. L., and Frommer, M. (1994) High sensitivity mapping of methylated cytosines, Nucleic Acids Res 22, 2990–2997.
Kuo, K. C., McCune, R. A., Gehrke, C. W., Midgett, R., and Ehrlich, M. (1980) Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA, Nucleic Acids Res 8, 4763–4776.
Gehrke, C. W., McCune, R. A., Gama-Sosa, M. A., Ehrlich, M., and Kuo, K. C. (1984) Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA, J Chromatogr 301, 199–219.
Eick, D., Fritz, H. J., and Doerfler, W. (1983) Quantitative determination of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography, Anal Biochem 135, 165–171.
Rubin, R. A., and Modrich, P. (1977) EcoRI methylase. Physical and catalytic properties of the homogeneous enzyme, J Biol Chem 252, 7265–7272.
Hubscher, U., Pedrali-Noy, G., Knust-Kron, B., Doerfler, W., and Spadari, S. (1985) DNA methyltransferases: activity minigel analysis and determination with DNA covalently bound to a solid matrix, Anal Biochem 150, 442–448.
Jeltsch, A., Friedrich, T., and Roth, M. (1998) Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase, J Mol Biol 275, 747–758.
Roth, M., and Jeltsch, A. (2000) Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases, Biol Chem 381, 269–272.
Liebert, K., and Jeltsch, A. (2008) Detection and quantitation of the activity of DNA methyltransferases using a biotin/avidin microplate assay, Methods Mol Biol 418, 149–156.
Grunau, C., Clark, S. J., and Rosenthal, A. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters, Nucleic Acids Res 29, E65-65.
Zhang, Y., Rohde, C., Tierling, S., Stamerjohanns, H., Reinhardt, R., Walter, J., and Jeltsch, A. (2009) DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones, Methods Mol Biol 507, 177–187.
Laird, C. D., Pleasant, N. D., Clark, A. D., Sneeden, J. L., Hassan, K. M., Manley, N. C., Vary, J. C., Jr., Morgan, T., Hansen, R. S., and Stoger, R. (2004) Hairpin-bisulfite PCR: assessing epigenetic methylation patterns on complementary strands of individual DNA molecules, Proc Natl Acad Sci U S A 101, 204–209.
Jurkowska, R. Z., Anspach, N., Urbanke, C., Jia, D., Reinhardt, R., Nellen, W., Cheng, X., and Jeltsch, A. (2008) Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L, Nucleic Acids Res 36, 6656–6663.
Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J Biomol Screen 4, 67–73.
Rohde, C., Zhang, Y., Reinhardt, R., and Jeltsch, A. (2010) BISMA--fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences, BMC Bioinformatics 11, 230.
Rohde, C., Zhang, Y., Jurkowski, T. P., Stamerjohanns, H., Reinhardt, R., and Jeltsch, A. (2008) Bisulfite sequencing Data Presentation and Compilation (BDPC) web server--a useful tool for DNA methylation analysis, Nucleic Acids Res 36, e34.
Rohde, C., Zhang, Y., Stamerjohanns, H., Hecher, K., Reinhardt, R., and Jeltsch, A. (2009) New clustering module in BDPC bisulfite sequencing data presentation and compilation web application for DNA methylation analyses, Biotechniques 47, 781–783.
Tusnady, G. E., Simon, I., Varadi, A., and Aranyi, T. (2005) BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes, Nucleic Acids Res 33, e9.
Li, L. C., and Dahiya, R. (2002) MethPrimer: designing primers for methylation PCRs, Bioinformatics 18, 1427–1431.
Yamada, Y., Watanabe, H., Miura, F., Soejima, H., Uchiyama, M., Iwasaka, T., Mukai, T., Sakaki, Y., and Ito, T. (2004) A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 21q, Genome Res 14, 247–266.
Jia, D., Jurkowska, R. Z., Zhang, X., Jeltsch, A., and Cheng, X. (2007) Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation, Nature 449, 248–251.
Friedhoff, P., Gimadutdinow, O., Ruter, T., Wende, W., Urbanke, C., Thole, H., and Pingoud, A. (1994) A procedure for renaturation and purification of the extracellular Serratia marcescens nuclease from genetically engineered Escherichia coli, Protein Expr Purif 5, 37–43.
Zhang, Y., Jurkowska, R., Soeroes, S., Rajavelu, A., Dhayalan, A., Bock, I., Rathert, P., Brandt, O., Reinhardt, R., Fischle, W., and Jeltsch, A. (2010) Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail, Nucleic Acids Res.
Handa, V., and Jeltsch, A. (2004) Anomalous mobility of polymerase chain reaction products after bisulfite treatment of DNA, Anal Biochem 333, 196–198.
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Jurkowska, R.Z., Ceccaldi, A., Zhang, Y., Arimondo, P.B., Jeltsch, A. (2011). DNA Methyltransferase Assays. In: Tollefsbol, T. (eds) Epigenetics Protocols. Methods in Molecular Biology, vol 791. Humana Press. https://doi.org/10.1007/978-1-61779-316-5_13
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DOI: https://doi.org/10.1007/978-1-61779-316-5_13
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