Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA.

Abstract

Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively.Analysis of substrate specificity with synthetic DNAs and restriction fragments of 4X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methylaccepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.DNA methylation in cells from vertebrates is a post-replication process involving the transfer of methyl groups from S-adenosyl L-methionine (AdoMet) to the 5 position of cytosine residues through the action of DNA methyltransferase [DNA MeTase; DNA (cytosine-5-)-methyltransferase, EC 2.1.1.37]. Methylated cytosine residues are located primarily within the dinucleotide 5' CpG 3' (1) and are found in tissue-specific amounts and positions (2). Sano and Sager (3) reported that, in bovine satellite I DNA, 5-methylcytosine (m5C) residues are located exclusively 5' to G residues, occur in clusters, and tend to be located within short self-complementary sequences; the distribution of m5C residues within this DNA showed striking tissue-specific variation.Changes in amounts and locations of m5C residues in DNA appear to be a part of the developmental program in higher cells (reviewed in ref. 4), and it has been demonstrated for many different genes that the loss of methyl groups from specific sites is correlated with the transcriptional activation of adjacent sequences (5-9). Also, in vitro enzymatic methylation of CpG dinucleotides by the bacterial restriction methyltransferase MHpa II inhibits expression of viral (10) and cellular (11) genes after introduction of the methylated DNA into cells. The pattern of methylation is maintained during subsequent cell divisions (12). Treatment of cultured cells with inhibitors of DNA methylation leads to the induction of certain genes with a concomitant reduction in the number of associated methylated CpG sites (13).Despite the apparent biological importance of DNA methylation it is not at all clear how patterns of methylation are established and maintained within the genome. DNA MeTases prepared from a number of rodent (14-17) and human (18) sources have been shown to require CpG-containing DNA, although additional sequence requirements have not been identified. A crude preparation of DNA MeTase from mouse ascites cells showed a strong pre...

Global delay in nascent strand DNA methylation

“…Three enzymes are generally responsible for establishing and maintaining this modification: DNA methyltransferases 1 (DNMT1), 3A (DNMT3A), and 3B (DNMT3B), all of which are essential for normal mammalian development 3 . Maintenance appears to be predominantly accomplished by DNMT1, which localizes to replication foci 4 and exhibits 10-40 fold higher binding affinity and catalytic activity towards hemi-methylated DNA substrates 5–7 . DNMT1 is also recruited to nascent DNA by the essential cofactor UHRF1 (ubiquitin-like, with PHD and RING finger domains 1), which exhibits a high affinity for hemi-methylated DNA through its SRA domain 8,9 and ubiquitinates the histone H3 tail to facilitate DNMT1 recruitment 10 .…”

Epigenetic mechanisms underlying the imprinting of the mouse H19 gene.

“…The parentalspecific methylation must be maintained through the period after fertilization when genome-wide demethylation of nonimprinted genes has been demonstrated (Monk et al 1987;Chaillet et al 1991;Kafri et al 1992). The difference could be maintained through many somatic cell divisions through the action of DNA methyltransferase, whose substrate is hemimethylated DNA (Gruenbaum et al 1982;Bestor and Ingram 1983). It could be erased in the next generation with the action of a DNA demethylase.…”

UHRF1‐mediated tumor suppressor gene inactivation in nonsmall cell lung cancer

“…9 DNMTs (DNA methyltransferases) are the key enzymes responsible for the establishment and maintenance of DNA methylation profiles. [10][11][12] Until recently, DNMT1 was considered to be the maintenance enzyme after DNA replication, whereas DNMT3A and DNMT3B were characterized as de novo methyltransferases. 13 The revised model suggests that DNMT1 carries out the bulk of the DNA methylation maintenance, whereas DNMT3A and DNMT3B, being compartmentalized to particular chromosome regions, can complete the methylation process and correct errors.…”