human dnmt1  (New England Biolabs)


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    Structured Review

    New England Biolabs human dnmt1
    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of <t>DNMT1</t> for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Human Dnmt1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation"

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00683

    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Figure Legend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Techniques Used: Binding Assay, Methylation

    2) Product Images from "Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation"

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00683

    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Figure Legend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Techniques Used: Binding Assay, Methylation

    3) Product Images from "Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation"

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00683

    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Figure Legend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Techniques Used: Binding Assay, Methylation

    4) Product Images from "Epigenetic alterations associated with dexamethasone sodium phosphate through DNMT and TET in RPE cells"

    Article Title: Epigenetic alterations associated with dexamethasone sodium phosphate through DNMT and TET in RPE cells

    Journal: Molecular Vision

    doi:

    DEX exposure reduces DNMT enzymatic activity in vitro. Relative enzymatic activity of ( A ) DNA methyltransferases (DNMTs) and ( B ) DNMT1 from purified human DNMTs. Upon dexamethasone (DEX) exposure, the activity of the DNMTs was inhibited. (Data are shown as mean ± standard deviation [SD] from three independent experiments.) *p
    Figure Legend Snippet: DEX exposure reduces DNMT enzymatic activity in vitro. Relative enzymatic activity of ( A ) DNA methyltransferases (DNMTs) and ( B ) DNMT1 from purified human DNMTs. Upon dexamethasone (DEX) exposure, the activity of the DNMTs was inhibited. (Data are shown as mean ± standard deviation [SD] from three independent experiments.) *p

    Techniques Used: Activity Assay, In Vitro, Purification, Standard Deviation

    5) Product Images from "Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *"

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.525279

    Cellular location and colocalization of PHF20L1a. A , the top panel shows pericentric localization of PHF20L1a. DNA staining is DAPI. In the middle panel , 5mC is shown in green and DNMT1K142me1 in red . In the bottom panel are shown CFP-SET7 in blue along with PHF20L1a in green and DNMT1K142me1 in red . The merge panel of DNMT1K142me1, PHF20L1a, and SET7 shows circular pericentric heterochromatin matching to the bright field image. Percentage perinucleolar localization was calculated from 200 transfected cells. B , colocalization of endogenous DNMT1, PHF20L1, and 5-methylcytosine in pericentric heterochromatin in HeLa cells. DAPI staining shows the heterochromatic regions.
    Figure Legend Snippet: Cellular location and colocalization of PHF20L1a. A , the top panel shows pericentric localization of PHF20L1a. DNA staining is DAPI. In the middle panel , 5mC is shown in green and DNMT1K142me1 in red . In the bottom panel are shown CFP-SET7 in blue along with PHF20L1a in green and DNMT1K142me1 in red . The merge panel of DNMT1K142me1, PHF20L1a, and SET7 shows circular pericentric heterochromatin matching to the bright field image. Percentage perinucleolar localization was calculated from 200 transfected cells. B , colocalization of endogenous DNMT1, PHF20L1, and 5-methylcytosine in pericentric heterochromatin in HeLa cells. DAPI staining shows the heterochromatic regions.

    Techniques Used: Staining, Transfection

    PHF20L1a inhibits DNMT1K142me1 ubiquitination and degradation. A , FLAG-PHF20L1a overexpression prevents ubiquitination of DNMT1K142me1. Overexpression ( O/E ) of GFP, GFP-SET7, FLAG-PHF20L1a, or a combination of GFP-SET7 and FLAG-PHF20L1a are indicated at the top of the Western blot. Overexpression of DsRed-DNMT1 and HA-ubiquitin and subsequent treatment with the MG132 proteasome inhibitor were carried out for all samples. Immunoprecipitations ( IP ) with anti-DNMT1 antibody were subsequently performed, and the precipitate was analyzed by Western blot. Input was revealed with anti-PHF20L1a, anti-SET7, anti-DsRed-DNMT1, anti-DNMT1K142me1, and anti-histone H3 antibodies, whereas DNMT1 immunoprecipitations were revealed with anti-HA-ubiquitin antibody (indicated on the right ). Full-length HA-ubiquitinated DNMT1 was present (*), but smaller degradation products were prevalent. Densitometry measurements of anti-HA-ubiquitin, normalized to histone H3, were done within the indicated brackets to show levels of ubiquitinated full-length DNMT1 (arbitrary units). B , loss of chromatin-bound DNMT1 in response to PHF20L1 knockdown is mediated by proteasomal degradation. siRNA-mediated knockdown of PHF20L1 and subsequent treatment with the MG132 proteasomal inhibitor are indicated at the top of the Western blot. Anti-PHF20L1a, anti-DNMT1, and anti-histone H3 antibodies used in the Western blot are indicated to the right of the blot. C , LC-MS analysis reveals that siRNA-mediated knockdown of PHF20L1 significantly reduces global levels of 5-methylcytosine. Genomic DNA was digested to single nucleosides for LC-MS analysis. Methylation changes were normalized to total levels of corresponding nucleotides. Total levels of 5-methylcytosine were significantly lower ( p = 0.029) after transfection of PH20L1 siRNA compared with control siRNA. Data represent two experimental replicates and four technical replicates. Error bars , S.D.
    Figure Legend Snippet: PHF20L1a inhibits DNMT1K142me1 ubiquitination and degradation. A , FLAG-PHF20L1a overexpression prevents ubiquitination of DNMT1K142me1. Overexpression ( O/E ) of GFP, GFP-SET7, FLAG-PHF20L1a, or a combination of GFP-SET7 and FLAG-PHF20L1a are indicated at the top of the Western blot. Overexpression of DsRed-DNMT1 and HA-ubiquitin and subsequent treatment with the MG132 proteasome inhibitor were carried out for all samples. Immunoprecipitations ( IP ) with anti-DNMT1 antibody were subsequently performed, and the precipitate was analyzed by Western blot. Input was revealed with anti-PHF20L1a, anti-SET7, anti-DsRed-DNMT1, anti-DNMT1K142me1, and anti-histone H3 antibodies, whereas DNMT1 immunoprecipitations were revealed with anti-HA-ubiquitin antibody (indicated on the right ). Full-length HA-ubiquitinated DNMT1 was present (*), but smaller degradation products were prevalent. Densitometry measurements of anti-HA-ubiquitin, normalized to histone H3, were done within the indicated brackets to show levels of ubiquitinated full-length DNMT1 (arbitrary units). B , loss of chromatin-bound DNMT1 in response to PHF20L1 knockdown is mediated by proteasomal degradation. siRNA-mediated knockdown of PHF20L1 and subsequent treatment with the MG132 proteasomal inhibitor are indicated at the top of the Western blot. Anti-PHF20L1a, anti-DNMT1, and anti-histone H3 antibodies used in the Western blot are indicated to the right of the blot. C , LC-MS analysis reveals that siRNA-mediated knockdown of PHF20L1 significantly reduces global levels of 5-methylcytosine. Genomic DNA was digested to single nucleosides for LC-MS analysis. Methylation changes were normalized to total levels of corresponding nucleotides. Total levels of 5-methylcytosine were significantly lower ( p = 0.029) after transfection of PH20L1 siRNA compared with control siRNA. Data represent two experimental replicates and four technical replicates. Error bars , S.D.

    Techniques Used: Over Expression, Western Blot, Liquid Chromatography with Mass Spectroscopy, Methylation, Transfection

    6) Product Images from "A real-time assay for CpG-specific cytosine-C5 methyltransferase activity"

    Article Title: A real-time assay for CpG-specific cytosine-C5 methyltransferase activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq047

    Break light cytosine-C5 methyltransferase activity assay. The fluorescence of the hemimethylated oligonucleotide 1 is quenched by the dabcyl group. It is a substrate for cytosine-C5 methyltransferases such as DNMT1 or M.SssI and upon methylation yields the fully methylated oligonucleotide 2 , which is cleaved by GlaI, separating the fluorophore from the quencher and resulting in an increase in fluorescence, which is proportional to the concentration of oligonucleotide 3 .
    Figure Legend Snippet: Break light cytosine-C5 methyltransferase activity assay. The fluorescence of the hemimethylated oligonucleotide 1 is quenched by the dabcyl group. It is a substrate for cytosine-C5 methyltransferases such as DNMT1 or M.SssI and upon methylation yields the fully methylated oligonucleotide 2 , which is cleaved by GlaI, separating the fluorophore from the quencher and resulting in an increase in fluorescence, which is proportional to the concentration of oligonucleotide 3 .

    Techniques Used: Activity Assay, Fluorescence, Methylation, Concentration Assay

    Activity of DNMT1 with oligonucleotide 1 . ( A ) Comparison of fluorescence changes in a full assay (solid black line) and a negative control assay lacking DNMT1 (dotted line). ( B ) Plot of change in the concentration of oligonucleotide 3 after background subtraction.
    Figure Legend Snippet: Activity of DNMT1 with oligonucleotide 1 . ( A ) Comparison of fluorescence changes in a full assay (solid black line) and a negative control assay lacking DNMT1 (dotted line). ( B ) Plot of change in the concentration of oligonucleotide 3 after background subtraction.

    Techniques Used: Activity Assay, Fluorescence, Negative Control, Concentration Assay

    Effect of increasing oligonucleotide 1 concentration on DNMT1 activity. ( A ) Real-time data averaged from duplicate assays. Assays contained oligonucleotide 1 at concentrations of 30 nM (black line), 60 nM (orange line), 120 nM (green line) and 240 nM (blue line) with associated negative controls (lacking DNMT1) shown as dotted lines. ( B ) Plot of steady-state rate against concentration of oligonucleotide 1 .
    Figure Legend Snippet: Effect of increasing oligonucleotide 1 concentration on DNMT1 activity. ( A ) Real-time data averaged from duplicate assays. Assays contained oligonucleotide 1 at concentrations of 30 nM (black line), 60 nM (orange line), 120 nM (green line) and 240 nM (blue line) with associated negative controls (lacking DNMT1) shown as dotted lines. ( B ) Plot of steady-state rate against concentration of oligonucleotide 1 .

    Techniques Used: Concentration Assay, Activity Assay

    7) Product Images from "Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro"

    Article Title: Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.3.704-723.2002

    GST-3a methylates circular plasmid DNA as well as linear DNA fragments in vitro using a restriction enzyme digestion assay. (A) Purified GST fusion protein of Dnmt3a wild type (GST-3a) and mutant (GST-3aMut). Six units of the DNMT1 (New England Biolabs) and approximately 200 ng each of GST-3a and GST3aMut were loaded on an SDS-7% polyacrylamide gel and stained with Coomassie blue stain. (B) Plasmid p220.0 was incubated (+) with GST-3a or GST3aMut, digested with a 10-fold excess of Hin P1I, and labeled with [ 32 P]dCTP using Klenow enzyme as described in Materials and Methods. The complete digestion pattern is shown in the lane that has no protein treatment. The additional bands in the lanes with DNA treated with GST-3a are the Hin P1I-resistant bands, indicating DNA methylation. These additional bands are marked by arrowheads. (C) A 467-bp DNA fragment containing three Hha I sites was used as a substrate for GST-3a and GST-3aMut, as described above. The DNA was digested with Hha I before being fractionated on a 1% agarose gel, Southern transferred, and probed with the entire DNA fragment. DNA unmethylated at the Hha I sites gives rise to the 304-bp band after Hha I digestion. The 467-bp band indicates methylation at the three Hha I sites.
    Figure Legend Snippet: GST-3a methylates circular plasmid DNA as well as linear DNA fragments in vitro using a restriction enzyme digestion assay. (A) Purified GST fusion protein of Dnmt3a wild type (GST-3a) and mutant (GST-3aMut). Six units of the DNMT1 (New England Biolabs) and approximately 200 ng each of GST-3a and GST3aMut were loaded on an SDS-7% polyacrylamide gel and stained with Coomassie blue stain. (B) Plasmid p220.0 was incubated (+) with GST-3a or GST3aMut, digested with a 10-fold excess of Hin P1I, and labeled with [ 32 P]dCTP using Klenow enzyme as described in Materials and Methods. The complete digestion pattern is shown in the lane that has no protein treatment. The additional bands in the lanes with DNA treated with GST-3a are the Hin P1I-resistant bands, indicating DNA methylation. These additional bands are marked by arrowheads. (C) A 467-bp DNA fragment containing three Hha I sites was used as a substrate for GST-3a and GST-3aMut, as described above. The DNA was digested with Hha I before being fractionated on a 1% agarose gel, Southern transferred, and probed with the entire DNA fragment. DNA unmethylated at the Hha I sites gives rise to the 304-bp band after Hha I digestion. The 467-bp band indicates methylation at the three Hha I sites.

    Techniques Used: Plasmid Preparation, In Vitro, Purification, Mutagenesis, Staining, Incubation, Labeling, DNA Methylation Assay, Agarose Gel Electrophoresis, Methylation

    8) Product Images from "Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine"

    Article Title: Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine

    Journal: Oncotarget

    doi:

    Decitabine reverses genome-wide DNA methylation and induces expression of genes associated with differentiation in IDH mutant glioma cells A, Low dose DAC inhibits DNMT1. Results from western blot shown. B, DAC treatment results in loss of DNA methylation. DNA methylome analysis of TS603 and TS667 cells following DAC treatment (200nM) is shown. Results from the Illumina HumanMethylation450 array. C, Gene expression changes following DAC treatment (200nM). Results from Affymetrix gene expression arrays. Most significantly altered genes following 200nM DAC are shown. D, Significant concordance between demethylated and upregulated genes and polycomb targets. Venn diagram showing overlap between the gene sets. P value (hypergeometric) is shown.
    Figure Legend Snippet: Decitabine reverses genome-wide DNA methylation and induces expression of genes associated with differentiation in IDH mutant glioma cells A, Low dose DAC inhibits DNMT1. Results from western blot shown. B, DAC treatment results in loss of DNA methylation. DNA methylome analysis of TS603 and TS667 cells following DAC treatment (200nM) is shown. Results from the Illumina HumanMethylation450 array. C, Gene expression changes following DAC treatment (200nM). Results from Affymetrix gene expression arrays. Most significantly altered genes following 200nM DAC are shown. D, Significant concordance between demethylated and upregulated genes and polycomb targets. Venn diagram showing overlap between the gene sets. P value (hypergeometric) is shown.

    Techniques Used: Genome Wide, DNA Methylation Assay, Expressing, Mutagenesis, Western Blot

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    New England Biolabs human dnmt1
    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of <t>DNMT1</t> for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Human Dnmt1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human dnmt1/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human dnmt1 - by Bioz Stars, 2022-07
    90/100 stars
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    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Journal: Biochemistry

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    doi: 10.1021/acs.biochem.8b00683

    Figure Lengend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Article Snippet: [ ] [ ] [ ] (8) Pradhan M, Esteve PO, Chin HG, Samaranayke M, Kim GD, and Pradhan S (2008) CXXC domain of human DNMT1 is essential for enzymatic activity .

    Techniques: Binding Assay, Methylation

    DEX exposure reduces DNMT enzymatic activity in vitro. Relative enzymatic activity of ( A ) DNA methyltransferases (DNMTs) and ( B ) DNMT1 from purified human DNMTs. Upon dexamethasone (DEX) exposure, the activity of the DNMTs was inhibited. (Data are shown as mean ± standard deviation [SD] from three independent experiments.) *p

    Journal: Molecular Vision

    Article Title: Epigenetic alterations associated with dexamethasone sodium phosphate through DNMT and TET in RPE cells

    doi:

    Figure Lengend Snippet: DEX exposure reduces DNMT enzymatic activity in vitro. Relative enzymatic activity of ( A ) DNA methyltransferases (DNMTs) and ( B ) DNMT1 from purified human DNMTs. Upon dexamethasone (DEX) exposure, the activity of the DNMTs was inhibited. (Data are shown as mean ± standard deviation [SD] from three independent experiments.) *p

    Article Snippet: For quantification of DNMT1 activity, assays were performed similarly with 1.5 unit DNMT1 (NEB, M0230L) with 1.5 h reaction time.

    Techniques: Activity Assay, In Vitro, Purification, Standard Deviation