DNA binding and methyl transfer catalysed by mouse DNA methyltransferase (original) (raw)
Related papers
Influence of Pre-existing Methylation on the de Novo Activity of Eukaryotic DNA Methyltransferase †
Biochemistry, 1998
Aberrant de novo methylation of CpG island DNA sequences has been observed in cultured cell lines or upon malignant transformation, but the mechanisms underlying this phenomenon are poorly understood. Using eukaryotic DNA (cytosine-5)-methyltransferase (of both human and murine origin), we have studied the in vitro methylation pattern of three CpG islands. Such sequences are intrinsically poor substrates of the enzyme, yet are efficiently methylated when a small amount of 5-methylcytosine is randomly introduced by the M.SssI prokaryotic DNA (cytosine-5)-methyltransferase prior to in vitro methylation by the eukaryotic enzyme. A stimulation was also found with several other double-stranded DNA substrates, either natural or of synthetic origin, such as poly(dG-dC)‚poly(dG-dC). An A+T-rich plasmid, pHb 1S, showed an initial stimulation, followed by a severe inhibition of the activity of DNA (cytosine-5)-methyltransferase. Methylation of poly(dI-dC)‚poly(dI-dC) was instead inhibited by preexisting 5-methylcytosines. The extent of stimulation observed with poly(dG-dC)‚poly(dG-dC) depends on both the number and the distribution of the 5-methylcytosine residues, which probably must not be too closely spaced for the stimulatory effect to be exerted. The activity of the M.SssI prokaryotic DNA methyltransferase was not stimulated, but was inhibited by pre-methylation on either poly(dG-dC)‚poly-(dG-dC) or poly(dI-dC)‚poly(dI-dC). The prokaryotic and eukaryotic DNA methyltransferases also differed in sensitivity to poly(dG-m 5 dC)‚poly(dG-m 5 dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.
Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases
Nucleic Acids Research, 2001
We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K m AdoMet , for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 µM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K m DNA values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 µM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 µM when poly(dG-dC)poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT ≥ CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.
Isolation and Expression of a Chicken DNA Methyltransferase cDNA^1
The Journal of Biochemistry, 1995
A 0.5 kb fragment of chicken DNA methyltransferase cDNA was PCR-amplified using a set of degenerate primers. A clone harboring a 5 kb insert was isolated from a cDNA library by screening with the PCR-amplified cDNA fragment as a probe. The elucidated nucleotide sequence gave a 4,614 nucleotide open reading frame, and the predicted protein was highly homologous to the mouse and human DNA methyltransferases, especially in the amino acid sequence of the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and Lys-Gly repeat first found in the mouse sequence were also conserved in chicken. However, about 250 amino acid residues in the amino-terminal portion of chicken DNA methyltransferase diverged from the amino-terminus of the mouse or human sequence.
Journal of Molecular Biology, 1997
The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-speci®c DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identi®ed. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identi®able de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not ®t the de®nition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence speci®city of intact Dnmt1 in whole-cell lysates. Speci®city was found to be con®ned to the sequence 5 H -CpG-3 H ; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-speci®c de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.
J Mol Biol, 1997
The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-speci®c DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identi®ed. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identi®able de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not ®t the de®nition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence speci®city of intact Dnmt1 in whole-cell lysates. Speci®city was found to be con®ned to the sequence 5 H -CpG-3 H ; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-speci®c de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.
DNA (cytosine-5)-methyltransferases in mouse cells and tissues. studies with a mechanism-based probe
Journal of Molecular Biology, 1997
The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-speci®c DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identi®ed. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identi®able de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not ®t the de®nition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence speci®city of intact Dnmt1 in whole-cell lysates. Speci®city was found to be con®ned to the sequence 5 H -CpG-3 H ; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-speci®c de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.
Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1987
The activity of eukaryotic DNA methyitransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 pg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) • poly(dG-dC) and poly(dA-dT) • poly(dA-dT), but not poly(dl-dC) • poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.
Biochemistry and biology of mammalian DNA methyltransferases
DNA methylation is a stable but not irreversible epigenetic signal that silences gene expression. It has a variety of important functions in mammals, including control of gene expression, cellular differentiation and development, preservation of chromosomal integrity, parental imprinting and X-chromosome inactivation. In addition, it has been implicated in brain function and the development of the immune system.
Nucleic acids …, 2002
DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of in¯uencing this process in vivo is therefore of signi®cant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc ®nger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate speci®c DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc ®nger subsites, targeted M.HpaII being the most speci®c. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc ®nger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are speci®c for the target site up to separations of~40 bp between the zinc ®nger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a signi®cant non-targeted activity most evident at elevated protein concentrations.
Purification and biochemical characterization of the Ecal DNA methyltransferase
European Journal of Biochemistry, 1992
The EcaI GGTNACC-specific DNA-adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25°C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The K, was 170 nM for DNA and 1.8 pM for the methyl donor S-adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki = 3.5 nM). The other product of the DNA-methylation reaction, S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine (Ki = 2.7 pM). The Sadenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.