Substrate preferences of human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides

Carotti, Daniela; Palitti, Franco; Mastrantonio, Stefania; Rispoli, Matilde; Strom, Roberto; Amato, Antonio; Campagnari, Francesco; Whitehead, Edward P.

doi:10.1016/0167-4781(86)90110-7

Cited by 19 publications

(14 citation statements)

References 25 publications

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“…This was surprising, as we had previously shown that singlestranded oligonucleotides did not accept methyl groups, although we had not investigated the accepting activities of all of the single-stranded oligonucleotides used. On the other hand, Carotti et al [9] had concluded that some random sequence polynucleotides could act as methyl group acceptors and, while the present work was in progress, Christman et al [10] reported similar findings. Moreover, Smith et al [11] have demonstrated that mispaired cytosines show enhanced accepting ability.…”

Section: Introductionsupporting

confidence: 65%

Spreading of methylation along DNA

Lindsay

Adams

1996

Biochemical Journal

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Mouse DNA methyltransferase is able to catalyse the transfer of a methyl group to certain CG-containing single-stranded oligonucleotides. The presence of a methylcytosine is required for efficient transfer. This methylcytosine may or may not be on the same oligonucleotide as that containing the accepting CG dinucleotide. When the accepting CG dinucleotide forms part of an unmethylated CG dinucleotide pair, its accepting activity is dramatically reduced. This provides the potential for methylation to spread along the DNA when it is rendered single-stranded at replication. It could also help to maintain fully methylated CG islands and asymmetrically methylated sites.

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Section: Introductionsupporting

confidence: 65%

Spreading of methylation along DNA

Lindsay

Adams

1996

Biochemical Journal

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“…The radioactivity was then measured in a Beckman LS6800 liquid scintillation spectrometer. Previous experiments [33] had shown that with ds-DNA the reaction is linear for at least 80 min at 37°C.…”

Section: Separation and Analysis Of Hi Histone Variantsmentioning

confidence: 94%

“…DNA methyltransferase assay DNA methyltransferase (EC 2.1.1.37) was purified from human placenta nuclei [33] and assayed according to Caiafa et al [34] in a 50 mM Tris/HCl buffer (pH 7.8) containing 10 % (v/v) glycerol, 5 mM EDTA, 0.5 mM DTT, 10 Ci/ml S-adenosyl-L-[methyl-3H]methionine (New England Nuclear; specific activity 70 Ci/mmol-80 Ci/mmol) and 30 ,g/ml of either Micrococcus luteus DNA or synthetic ds-oligonucleotides. Assay mixtures were incubated at 37°C for 1 h and the reaction was terminated by heating to 60°C for 20 min in the presence of 1 % (w/v) SDS and 0.2 ,tg/ml of proteinase K. After cooling on ice, 300 ,ug of salmon sperm DNA was added to serve as carrier and the DNA was precipitated at 0°C with 10% (w/v, final concentration) trichloroacetic acid.…”

Section: Separation and Analysis Of Hi Histone Variantsmentioning

confidence: 99%

Binding of histone H1e-c variants to CpG-rich DNA correlates with the inhibitory effect on enzymic DNA methylation

Santoro

D’Erme²,

Mastrantonio

et al. 1995

Biochemical Journal

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Within the H1 histone family, only some fractions enriched in the H1e-c variants are effective in causing a marked inhibition, in vitro, of enzymic DNA methylation and, in gel retardation and Southwestern blot experiments, in binding double-stranded (ds) CpG-rich oligonucleotides. Both the 6-CpG ds-oligonucleotide and the DNA purified from chromatin fractions enriched in 'CpG islands' are good competitors for the binding of H1e-c to 6-meCpG ds-oligonucleotide. Because of their ability to bind any DNA sequence and to suppress the enzymic methylation in any sequence containing CpG dinucleotides, these particular H1 variants could play some role in maintaining linker DNA at low methylation levels and even in preserving the unmethylated state of the CpG-rich islands which characterize the promoter regions of housekeeping genes.

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“…Steady-state Kinetic Properties of Recombinant Human DNA (Cytosine-5) Methyltransferase on Poly(dI-dC)⅐Poly(dI-dC) Substrate-Historically, poly(dI-dC)⅐poly(dI-dC) has been used to define the biochemical properties of mammalian m 5 C MTases (36,37). Each molecule of poly(dI-dC)⅐poly(dI-dC) provides a large number of potential (CI) dinucleotide sites for methylation.…”

Section: Expression and Purification Of The Biologically Activementioning

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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Substrate preferences of human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides

Cited by 19 publications

References 25 publications

Spreading of methylation along DNA

Spreading of methylation along DNA

Binding of histone H1e-c variants to CpG-rich DNA correlates with the inhibitory effect on enzymic DNA methylation

Recombinant Human DNA (Cytosine-5) Methyltransferase

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