Advertisement

Measuring DNA Demethylase Activity In Vitro

Protocol
  • 946 Downloads
Part of the Methods in Molecular Biology™ book series (MIMB, volume 200)

Abstract

The reaction catalyzing direct demethylation of DNA involves the removal of a methyl group residue from the 5′ position on cytosine; the products of the reaction are nonmethylated cytosine in the dinucleotide CpG and methanol (1). The study of the proteins involved in demethylation requires an assay for measuring enzymatic DNA demethylation. A number of assays were previously described for determining the state of methylation of CpG sequences DNA. For example, certain restriction enzymes such as HpaII or HhaI recognize subsets of CpG sequences only when the C is not methylated; thus, cleavage by this enzymes indicates demethylation of their recognition sequences (2) This assay is, however, obviously indirect and can only measure the state of methylation of a subset of CG sequences contained in the enzyme-specific recognition site. An additional problem is that this assay does not differentiate between DNA that is directly demethylated, and repair processes that remove methylated cytosines in DNA and replace them with other unmethylated cytosines found in the extracts. An additional assay is the bisulfite-mapping method, which can determine the state of methylation of cytosines at a single nucleotide resolution (3). This assay is based on the fact that nonmethylated cytosines are modified by bisulfite and converted to thymidine, whereas methylated cytosines are protected. This assay, similar to the restriction enzyme-based assays, is indirect; it does not measure demethylation but rather the conversion of cytosines.

Keywords

Isobutyric Acid Micrococcal Nuclease Unmethylated Cytosine Single Nucleotide Resolution Nap5 Column 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Ramchandani, S., Bhattacharya, S. K., Cervoni, N., and Szyf, M. (1999) DNA methylation is a reversible biological signal. Proc. Natl. Acad. Sci. USA 96, 6107–6112.PubMedCrossRefGoogle Scholar
  2. 2.
    Waalwijk, C. and Flavell, R. A. (1978) DNA methylation at a CCGG sequence in the large intron of the rabbit b-globin gene: tissue specific variations. Nucleic Acids. Res. 5,4631–4641.PubMedCrossRefGoogle Scholar
  3. 3.
    Clark, S. J., Harrison, C. L., Paul, C. L., and Frommer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22 2990–2997.PubMedCrossRefGoogle Scholar
  4. 4.
    Szyf, M. Theberge, J., and Bozovic, V. (1995) Ras induces a general DNA demethylation activity in mouse embryonal P19 cells. J.Biol. Chem. 270,12,690–12,696.PubMedCrossRefGoogle Scholar
  5. 5.
    Bhattacharya, S. K., Ramchandani, S., Cervoni, N., and Szyf, M. (1999) A mammalian protein with specific demethylase activity for mCpG DNA. Nature 397 579–583.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  1. 1.Department of Pharmacology and TherapeuticsMcGill UniversityMontrealCanada

Personalised recommendations