Influence of Pre-existing Methylation on the de Novo Activity of Eukaryotic DNA Methyltransferase † (original) (raw)
Related papers
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.
Biomedicine & Pharmacotherapy, 1992
Methylation of C residues at specific sites in DNA of higher eukaryotes appears to play a role in regulating gene expression during development and differentiation. Silencing of genes by methylation can in part explain genomic imprinting and alterations in patterns of methylation during the early stages of carcinogenesis may account for some changes in gene expression in tumor cells [l]. Although the distribution of 5mC in mammalian DNA is tissue-and species-specific, little is known about MTases role in establishing specific patterns of methylation. Mammalian DNA (mDNA)MTase is most active in methylating C residues in hemi-methylated CpG sites and prefers substrates with high GC content and multiple CpGs-15 residues apart [21. To more fully characterize the specificity of mDNA MTase, we analyzed its activity with a variety of defined DNA substrates and compared it with a bacterial MTase, Sssi, which is reported to act on hemi-methylated and unmethylated CpG sites with equal efficiency [3]. Using defined unmethylated single(ss)-and double stranded(DNAs as substfates, we find that: 1) the initial rate of methylation of both ss-and ds-DNA-by Sssl and mDNA MTase is influenced by sequence, often in opposite ways; 2) depending on sequence, Sssf may be more active on ds-than ss-DNA. Activity of mDNA Mtase with ss-DNA is highly variable and dependant on sequence but is always low with unmethylated ds-DNA. Using SmC-substituted substrates, we find that hemi-methylation either has no effect or inhibits activity of Sssl but always activates mDNA MTase. mDNA MTase is most active with hemi-methylated ds-DNA and ss-DNA with 5mC near the 5'-end. These results demonstrate that although both MTases catalyze the same reaction, methylation of C residues in CpG dinucleotides. they have quite different specificities. This should be considered when these enzymes are used to quantitate hypomethylation at CpG sites in DNA resulting from exposure of mammalian cells or tissues to conditions or agents that affect DNA methylation. Finally, our findings suggest that 5mC residues in specific sequences in ss-DNA may serve to activate de novo methylation in mammalian cells allowing alteration of existing methylation patterns.
DNA methylation pattern is determined by the intracellular level of the methylase
Proceedings of the National Academy of Sciences, 1984
Extrachromosomal plasmid DNA is transiently undermethylated in Escherichia coli during amplification in the presence of chloramphenicol. In addition, undermethylation of phage X DNA was observed after thermal induction of a XcI857 lysogen while the integrated X phage DNA was found to be fully methylated. These methylation pattern changes occur under conditions (extensive replication) in which the intracellular methylase level becomes limiting. In an E. coli strain that harbors a plasmid that carries the dam methylase gene and therefore overproduces dam methylase, there is no undermethylation of dam sites in either of the extrachromosomal DNAs. The sites that are methylated by the mec methylase in both plasmid and A phage DNAs were undermethylated in the dam overproducer as well. These results indicate that the intracellular level of the E. coli methylase determines the DNA methylation pattern.
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.
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.
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.
Gene, 2005
DNA methylation plays a central role in the control of gene expression during development and cell differentiation, thus DNA methylation and demethylation processes are expected to be strictly regulated during these events. We have explored the expression levels of the genes encoding DNA methylases, methyl-CpG binding proteins and demethylases during in vitro differentiation of human carcinoma colon cells (CaCO-2) used as a model system. The results show that the global DNA methylation pattern remains constant during CaCO-2 cells differentiation indicating that required genome methylation pattern in cell differentiation was already established in the seeded cells. On the contrary, the timing of expression of several of the explored genes is tightly regulated, suggesting they are involved in the regulation of the differentiation program. In particular, the timing of expression of DNMT3b and of MBD2b and 5-MCDG shows two peaks not observed in the time courses of the expression of other genes belonging to the same families. These events, not dependent on the cell cycle synchronization, have apparently no significant impact on the overall methylation status of the genome. D