Journal of Molecular Biology
Regular articleEnzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpA sites1
Introduction
Methylation of cytosine to 5-methylcytosine in the DNA of mammals is implicated in various biological and developmental processes (for reviews, see1, 2, 3, 4), such as gene regulation, regulation of the chromatin structure, DNA replication, parental imprinting, X-chromosome inactivation in females, embryonic development, carcinogenesis, and genetic diseases. Methylation occurs predominantly at CpG sites, 70–80 % of which are modified in mammalian DNA. However, significant amounts of 5-methylcytosine are also found at sites like CpNpG in plants5 or CpN in mammals.6 The modification is introduced after DNA replication by DNA methyltransferases (MTases), which transfer a methyl group from S-adenosylmethionine (AdoMet) to the 5-position of cytosine (for reviews, see7, 8, 9, 10, 11).
Modified and unmodified CpG sites create a specific pattern of methylation. Stable transmission of this pattern requires that after DNA replication, hemimethylated CpG sites are recognized and methylated by a maintenance MTase. In addition, creation of this pattern depends on selective de novo methylation and demethylation of certain regions of the DNA.12, 13 So far, three active DNA methyltransferases have been identified in mammals: Dnmt1, Dnmt3a and Dnmt3b. The Dnmt1 MTase has been known for several years and has been well studied (for reviews see9, 14). It has been shown to possess a high preference for methylation of hemimethylated CpG-sites in vitro,15, 16, 17, 18, 19 suggesting its involvement in maintenance methylation, a role that has also been confirmed by analysis of a Dnmt1−/− mouse.20 However, the de novo methylation of retroviral DNA persists in embryonic stem cells lacking Dnmt1, implying the presence of an additional MTase activity.21
The Dnmt3 MTases were discovered in 1998.22 Dnmt3a and 3b are heavily expressed in embryonic tissues, whereas only low expression is observed in differentiated cells.22, 23 The gene products comprise 908 and 776 amino acid residues, respectively, and share 36 % amino acid sequence identity (>60 % in the C-terminal half of the proteins). There are numerous alternative splice variants of Dnmt3b.22 Baculovirus-expressed Dnmt3a and Dnmt3b proteins were found to methylate CpG dinucleotides without preference for hemimethylated DNA.22 This feature makes them the best candidates for the de novo methylation function. Mutations in human Dnmt3B cause ICF, a severe, hereditary disease.24, 25, 26 Transgenic mice lacking Dnmt3a and Dnmt3b, singly and in combination, are hypomethylated and die at embryonic stages (Dnmt3a−/−/Dnmt3b−/−, Dnmt3b−/−) or shortly after birth (Dnmt3a−/−), indicating the critical role of these enzymes during development.26 De novo methylation of DNA by Dnmt3a and 3b was also demonstrated in vivo after expression in human cell lines27 and by expression of Dnmt3a in transgenic Drosophila melanogaster.28 However, the enzymatic properties of Dnmt3a or Dnmt3b have not been studied. Here, we present an in vitro study using recombinant Dnmt3a. We show that the enzymatic properties of Dnmt3a can be related directly to the specificity of de novo methylation in vivo. Furthermore, we suggest a model in which Dnmt1 and Dnmt3a cooperate in de novo methylation of DNA.
Section snippets
Cloning, protein expression, and in vivo activity of Dnmt3a
The gene for the murine Dnmt3a (GenBank accession no. NP_031898) was cloned into the expression vector pET28a at NdeI and HindIII restriction sites. The presence of the Dnmt3a gene in the expression plasmid was confirmed by PCR and restriction analysis. The cloned gene was sequenced completely to ensure the absence of mutations. The expression of the N-terminal His6-tag Dnmt3a fusion-protein was induced in transformed HSM174(DE3), BL21(DE3, pLysS) or XLBlue(MRF, DE3) E. coli cells. Dnmt3a
Oligodeoxynucleotides
HPLC-purified oligodeoxyribonucleotides were purchased from MWG (Ebersberg, Germany). The concentrations of oligonucleotide solutions were determined spectroscopically using E260 nm values provided by the manufacturer. Duplex oligonucleotides were prepared by adding equimolar amounts of complementary strands, heating to 95°C and slow cooling to room temperature. The following oligonucleotides were used, where M represents 5-methylcytosine and Bt represents biotin. Since all substrates have the
Acknowledgements
Many thanks are due to Dr E. Li (Charlestown, MA) for the gift of the cDNA of Dnmt3a and Dnmt3b. This work has been supported by the Deutsche Forschungsgemeinschaft (JE 252/1-1).
References (31)
Dynamics of DNA methylation pattern
Curr. Opin. Genet. Dev.
(2000)Methylation moves into medicine
Curr. Biol.
(2000)- et al.
CG and CNG methyltransferases in plants
Gene
(1995) DNA modification by methyltransferases
Curr. Opin. Struct. Biol.
(1995)- et al.
DNA methyltransferases
Curr. Opin. Cell Biol.
(1994) - et al.
Recombinant human DNA (cytosine-5) methyltransferase
J. Biol. Chem.
(1999) - et al.
Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated DNA
J. Biol. Chem.
(1999) - et al.
The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA
J. Mol. Biol.
(2001) - et al.
Targeted mutation of the DNA methyltransferase gene results in embryonic lethality
Cell
(1992) - et al.
DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development
Cell
(1999)
Molecular enzymology of the EcoRV DNA-(adenine-N6)-methyltransferasekinetics of DNA binding and bending, kinetic mechanism, and linear diffusion of the enzyme on DNA
J. Mol. Biol.
Cancer epigenetics comes of age
Nature. Genet.
Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G0/G1 to S phase transition in normal and tumor cells
Nucl. Acids Res.
Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a
Proc. Natl Acad. Sci. USA
Structure and function of DNA methyltransferases
Annu. Rev. Biophys. Biomol. Struct.
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