Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition

Adams, Roger; Gardiner, Katheleen; Rinaldi, A.; Bryans, Margaret; McGarvey, Margaret; Burdon, Roy H.

doi:10.1016/0167-4781(86)90080-1

Cited by 26 publications

(17 citation statements)

References 29 publications

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“…The DNA methylase from mealybug shows preferential methylation of denatured DNA even under low salt (50 mM) conditions. This is not a universal property of DNA methylases, but Adams et aL [28] have reported a similar phenomenon with DNA methylase purified from mouse ascites cells. The sequence specificity of the methylase as assessed by using synthetic polymers shows that both CpG as well as CpA dinucleotides are methylated by this enzyme.…”

Section: Discussionmentioning

confidence: 70%

“…This is referred to as the 'processive' mode of action [27]. A distinction between these two modes of action can be made by sequential addition of substrates differing in their methyl acceptor capacity [28]. In this study, the reaction was initiated with a DNA sample as the first substrate and after five minutes of incubation at 37 ° C, the second substrate was added.…”

Section: Mode Of Actionmentioning

confidence: 99%

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Detection of a CpA methylase in an insect system: characterization and substrate specificity

Devajyothi

Brahmachari

1992

Mol Cell Biochem

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A cytosine-specific DNA methyltransferase (EC 2.1.1.37) has been purified to near homogeneity from a mealybug (Planococcus lilacinus). The enzyme can methylate cytosine residues in CpG sequences as well as CpA sequences. The apparent molecular weight of the enzyme was estimated as 135,000 daltons by FPLC. The enzyme exhibits a processive mode of action and a salt dependence similar to mammalian methylases. Mealybug methylase exhibits a preference for denatured DNA substrates.

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Section: Discussionmentioning

confidence: 70%

Section: Mode Of Actionmentioning

confidence: 99%

Detection of a CpA methylase in an insect system: characterization and substrate specificity

Devajyothi

Brahmachari

1992

Mol Cell Biochem

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“…Previously, several purification attempts were described for mammalian m 5 C MTases using human placental tissue (37), murine erythroleukemia cells (13), and murine ascites cells (45). However, because of the small amount of the protein and the many purification steps required to approach homogeneity, it has been difficult to purify these proteins.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Human DNA (Cytosine-5) Methyltransferase

Pradhan¹,

Bacolla²,

Wells³

et al. 1999

Journal of Biological Chemistry

551

197

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A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces ϳ1 mg of intact recombinant enzyme >95% pure per 1.5 ؋ 10 9 insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two Cterminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dIdC)⅐poly(dI-dC) and unmethylated and hemimethylated 36-and 75-mer oligonucleotides. The turnover number (k cat ) was 131-237 h ؊1 on poly(dI-dC)⅐poly(dI-dC), 1.2-2.3 h ؊1 on unmethylated DNA, and 8.3-49 h ؊1 on hemimethylated DNA. The Michaelis constants for DNA (K m CG ) and S-adenosyl-L-methionine (AdoMet) (K m AdoMet ) ranged from 0.33-1.32 and 2.6 -7.2 M, respectively, whereas the ratio of k cat /K m CG ranged from 3.9 to 44 (237-336 for poly(dI-dC)⅐poly(dI-dC)) ؋ 10 6 M ؊1 h ؊1 . The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k cat on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.Methylated cytosine is found in the genome of organisms ranging from prokaryotes to mammals (1). Methylation of DNA in eukaryotes is implicated in various biological and developmental processes, such as gene regulation (2), DNA replication (3), genomic imprinting (4), embryonic development (5), carcinogenesis (6), and genetic diseases (7). The bulk of the methylation takes place during DNA replication in the S-phase of the cell cycle (8). The maintenance methylation ensures the propagation of tissue-specific methylation patterns established during mammalian development. The methyl transfer reaction proceeds via nonspecific binding of the enzyme to DNA, recognition of the specific DNA target site, and recruitment of the methyl group donor S-adenosyl-L-methionine (AdoMet) 1 to the active site of the enzyme. DNA (cytosine-5) methyltransferases (m 5 C MTase) introduce a methyl group onto carbon 5 of the target cytosine through a covalent intermediate between the protein and the target cytosine (9). During this process, the cytosine is flipped 180°out of the DNA backbone into an active site pocket of the enzyme (10). After completion of the methyl transfer reaction, the products, methylated DNA and S-adenosyl-L-homocysteine (AdoHcy), are released. Previous studies on the mechanism of methylation were mainly limited to prokaryotic m 5 C MTases although some limited kinetic studies have also been reported with mouse and human DNMT1 (11,12...

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“…More recently the purification of DNA methylase from wheat embryo has been reported (Theiss et al, 1987). This enzyme is reported to have a native Mr of 50000-55000, in contrast with the high-Mr enzyme from mammalian cells, which has an Mr value of 190000 for the intact enzyme (Pfeifer et al, 1985;Adams et al, 1986;Bestor et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

DNA methylase from Pisum sativum

Yesufu

Hanley

Rinaldi

et al. 1991

Biochemical Journal

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DNA methylase activity was detected in nuclei from pea shoots. The enzyme can only be extracted by low-salt treatment if the nuclei are pretreated with micrococcal nuclease. Only a single enzyme was detected, and it was purified to a specific activity of 1620 units/mg of protein. It has an Mr of 160,000 on gel filtration and SDS/PAGE. Pea DNA methylase methylates cytosine in all four dinucleotides, and this is interpreted to show that it acts on CNG trinucleotides. Although it shows a strong preference for hemi-methylated double-stranded DNA, it is also capable of methylation de novo. Homologous DNA is the best natural substrate. In vitro the enzyme interacts with DNA to form a salt-resistant complex with DNA that is stable for at least 4 h.

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Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition

Cited by 26 publications

References 29 publications

Detection of a CpA methylase in an insect system: characterization and substrate specificity

Detection of a CpA methylase in an insect system: characterization and substrate specificity

Recombinant Human DNA (Cytosine-5) Methyltransferase

DNA methylase from Pisum sativum

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