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S U P P O R T
 

Digestion of Methylated DNA

DNA methylation is the process of transfering a methyl group from a donor molecule to either a cytosine or to an adenine by DNA methyltransferases. Such methylation is the most common and abundant DNA modification process in living organisms. Three types of methylated bases are predominantly found in DNA:

5-methylcytosine (m5C),
N4-methylcytosine (m4C),
N6-methyladenine (m6A).

Other modified bases, such as 5-hydroxymethylcytosine (hm5C) and 5-hydroxymethyluracil (hm5U), have also been described. The organism-specific pattern of methylation depends on the methyltransferases’ specificity.
In prokaryotes, DNA cleavage by a cognate restriction enzyme is prevented by the methylation of DNA by a sequence-specific methyltransferase which is an integral component of every restriction-modification system (1, 2).
The majority of E.coli strains used for propagation of plasmid DNA contain two site-specific DNA methyltransferases - Dam and Dcm (3, 4). The methylase encoded by the dam gene methylates the N6-position of an adenine residue within the GATC sequence (5, 6). The methylase encoded by the dcm gene methylates the C5-position of an internal cytosine residue within the CCWGG sequence (4, 7).
In addition to Dam and Dcm methylases, laboratory strains of E.coli K12 and B may contain EcoKI or EcoBI enzymes, respectively, encoded by a type I R-M system. These methyltransferases modify adenine residues within their respective recognition sequences: AAC(N)6GTGC for EcoKI and TGA(N)8 TGCT for EcoBI (3, 4).
DNA from higher eukaryotic organisms possesses modified 5-methylcytosine residues within CpG or CpNpG contexts (2, 8-10). These tissue-specific methylation patterns are heritable. They participate in regulation of gene expression and cellular differentiation.
Most restriction enzymes are sensitive to DNA methylation. In the case of overlapping of a restriction enzyme target site with the methylation site, the following results are possible:

The ability to cleave methylated DNA is an intrinsic and unpredictable property of each restriction enzyme. Therefore, isoschizomers and neoschizomers which recognize the same DNA sequences can differ in their sensitivity to DNA methylation (see table below). For instance, MboI (recognition sequence GATC) does not cleave DNA methylated by Dam methylase (11), while the isoschizomer Bsp143I is insensitive to Dam methylation. Also, EcoRII does not cleave DNA at the CCWGG site if it is methylated by Dcm, while its neoisoschizomer MvaI will cleave this methylated site (12).
Thus, to achieve effective DNA digestion, it is necessary to take into account both the type of DNA methylation and the sensitivity of the restriction endonuclease to that type of methylation.
All restriction enzymes produced by Fermentas have been examined for their sensitivity to Dam, Dcm, CpG, EcoKI and EcoBI methylation of substrate DNA. Detailed information is presented in in the product descriptions and in the Certificates of Analysis supplied with each enzyme.

Table. Fermentas Isoschizomers and Neoschizomers with Differing Sensitivities to the Target Methylation.
Enzyme couple Recognition and
cleavage sites		 Sensitivity to methylation
Acc65I G^GTACC Overlapping Dcm or CpG methylation may influence DNA cleavage.
KpnI GGTAC^C Not influenced by Dcm or CpG methylation.
ApaI GGGCC^C Overlapping Dcm or CpG methylation may influence DNA cleavage.
Bsp120I G^GGCCC Blocked by overlapping Dcm or CpG methylation.
Bsp143I ^GATC Not influenced by Dam, blocked by CpG methylation.
MboI ^GATC Blocked by Dam methylated DNA.
DpnI GA^TC Cleaves only Dam methylated DNA.
Cfr9I C^CCGGG CpG methylation may influence DNA cleavage.
SmaI CCC^GGG Blocked by CpG methylation.
Csp6I G^TAC Not influenced by CpG methylation.
RsaI GT^AC Overlapping CpG methylation may influence DNA cleavage.
Ecl136II GAG^CTC Overlapping CpG methylation may influence DNA cleavage.
SacI GAGCT^C Not influenced by CpG methylation.
EcoRII ^CCWGG Blocked by Dcm methylation.
MvaI CC^WGG Not influenced by Dcm methylation.
HpaII C^CGG Blocked by CpG methylation.
MspI C^CGG Not influenced by CpG methylation.

References

  1. McClelland, M., The effect of sequence specific DNA methylation on restriction endonuclease cleavage, Nucleic Acids Res., 9, 5859-5866, 1981.
  2. McClelland, M., et al., Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases, Nucleic Acids Res., 22, 3640-3659, 1994.
  3. Marinus, M.G., Morris, N.R., Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12, J. Bacteriol., 114, 1143-1150, 1973.
  4. May, M.S., Hattman, S., Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes, J. Bacteriol., 123, 768-770, 1975.
  5. Hattman, S., et al., Sequence specificity of the P1 modification methylase (M.EcoP1) and the DNA methylase (M.Ecodam) controlled by the Escherichia coli dam gene, J. Mol. Biol.,126, 367-380, 1978.
  6. Geier, G.E., Modrich, P., Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of DpnI endonuclease, J. Biol. Chem., 254, 1408-1413, 1979.
  7. Buryanov, Ya.I., et al., Site specificity and chromatographic properties of E.coli K12 and EcoRII DNA-cytosine methylases, FEBS Letters, 88, 251-254, 1978.
  8. Waalwijk, C., Flavell, R.A., MspI, an isochizomer of HpaII which cleaves both unmethylated and methylated HpaII sites, Nucleic Acids Res., 5, 3231-3236, 1978.
  9. Bird, A.P., et al., Methylated and unmethylated DNA compartments in the sea urchin genome, Cell, 17, 889-902, 1979.
  10. McClelland, M., The frequency and distribution of methylatable DNA sequences in leguminous plant protein coding genes, J. Mol. Evol., 19, 346-354, 1983.
  11. Dreiseikelmann, B., et al., The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis, Biochim. Biophys. Acta, 562, 418-428, 1979.
  12. Butkus, V. et al., Investigation of restriction-modification enzymes from M.varians RFL19 with a new type of specificity toward modification of substrate, Nucleic Acids Res., 13, 5727-5746, 1985.
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Updated lapkričio 08, 2006 11:07