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 26 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 26 article reviews
    Price from $9.99 to $1999.99
    human dnmt1 - by Bioz Stars, 2022-08
    90/100 stars

    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 "Cyclophosphamide Perturbs Cytosine Methylation in Jurkat-T Cells through LSD1-mediated Stabilization of DNMT1 Protein"

    Article Title: Cyclophosphamide Perturbs Cytosine Methylation in Jurkat-T Cells through LSD1-mediated Stabilization of DNMT1 Protein

    Journal: Chemical research in toxicology

    doi: 10.1021/tx2003849

    Western blot results for DNMT1 (A), LSD1 (B), and Set7 (C) and DNMT1 with monomethylation on K142 (D) in the whole cell lysate of Jurkat-T cells treated with 10 µM cyclophosphamide for the indicated periods of time. The culture medium was changed
    Figure Legend Snippet: Western blot results for DNMT1 (A), LSD1 (B), and Set7 (C) and DNMT1 with monomethylation on K142 (D) in the whole cell lysate of Jurkat-T cells treated with 10 µM cyclophosphamide for the indicated periods of time. The culture medium was changed

    Techniques Used: Western Blot

    4) 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

    5) 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

    6) Product Images from "5-Aza-Deoxycytidine Induces Selective Degradation of DNA Methyltransferase 1 by a Proteasomal Pathway That Requires the KEN Box, Bromo-Adjacent Homology Domain, and Nuclear Localization Signal"

    Article Title: 5-Aza-Deoxycytidine Induces Selective Degradation of DNA Methyltransferase 1 by a Proteasomal Pathway That Requires the KEN Box, Bromo-Adjacent Homology Domain, and Nuclear Localization Signal

    Journal:

    doi: 10.1128/MCB.25.11.4727-4741.2005

    Mutation in the KEN box and deletions in the BAH domain or the NLS stabilize Dnmt1 against 5-aza-CdR-induced degradation, whereas mutation of cysteine in the PCQ motif cannot protect it from degradation. (A) Schematic representation of various mutants
    Figure Legend Snippet: Mutation in the KEN box and deletions in the BAH domain or the NLS stabilize Dnmt1 against 5-aza-CdR-induced degradation, whereas mutation of cysteine in the PCQ motif cannot protect it from degradation. (A) Schematic representation of various mutants

    Techniques Used: Mutagenesis

    5-Aza-CdR-induced degradation of Dnmt1 occurs in ts20 cells only when E1 is active at a permissive temperature (34°C). (A) 5-Aza-CdR-induced degradation is blocked in E1 mutant cells at a nonpermissive temperature. ts20 cells grown at 34°C
    Figure Legend Snippet: 5-Aza-CdR-induced degradation of Dnmt1 occurs in ts20 cells only when E1 is active at a permissive temperature (34°C). (A) 5-Aza-CdR-induced degradation is blocked in E1 mutant cells at a nonpermissive temperature. ts20 cells grown at 34°C

    Techniques Used: Mutagenesis

    5-Aza-CdR-induced degradation of DNMT1 remains unaffected in HeLa cells when DNA synthesis is blocked with aphidicolin. (A) BrdUrd incorporation is abolished in cells treated with aphidicolin. HeLa cells were treated with aphidicolin (20 μg/ml)
    Figure Legend Snippet: 5-Aza-CdR-induced degradation of DNMT1 remains unaffected in HeLa cells when DNA synthesis is blocked with aphidicolin. (A) BrdUrd incorporation is abolished in cells treated with aphidicolin. HeLa cells were treated with aphidicolin (20 μg/ml)

    Techniques Used: DNA Synthesis

    DNMT1 is selectively degraded by 5-aza-CdR treatment of mammalian cells. (A) Dnmt1, Dnmt3a, and Dnmt3b protein levels in P1798 cell extracts. Western blot analysis of Dnmt1/3a/3b with WCEs from mouse lymphosarcoma cells (100 μg of protein) treated
    Figure Legend Snippet: DNMT1 is selectively degraded by 5-aza-CdR treatment of mammalian cells. (A) Dnmt1, Dnmt3a, and Dnmt3b protein levels in P1798 cell extracts. Western blot analysis of Dnmt1/3a/3b with WCEs from mouse lymphosarcoma cells (100 μg of protein) treated

    Techniques Used: Western Blot

    5-Aza-CdR-induced degradation of DNMT1 occurs at the posttranslational level and can be blocked by proteasomal inhibitors. (A) Expression of DNMT1 mRNA is not reduced in cells upon treatment with 5-aza-CdR. The mRNA levels (copy numbers) of DNMTs normalized
    Figure Legend Snippet: 5-Aza-CdR-induced degradation of DNMT1 occurs at the posttranslational level and can be blocked by proteasomal inhibitors. (A) Expression of DNMT1 mRNA is not reduced in cells upon treatment with 5-aza-CdR. The mRNA levels (copy numbers) of DNMTs normalized

    Techniques Used: Expressing

    5-Aza-CdR-induced degradation of DNMT1 occurs in the nucleus and can be blocked by a proteasomal inhibitor (lactacystin). (A) Western blot analysis of DNMT1 in nuclear and cytoplasmic fractions. Cells were treated with 20 μM lactacystin for 1
    Figure Legend Snippet: 5-Aza-CdR-induced degradation of DNMT1 occurs in the nucleus and can be blocked by a proteasomal inhibitor (lactacystin). (A) Western blot analysis of DNMT1 in nuclear and cytoplasmic fractions. Cells were treated with 20 μM lactacystin for 1

    Techniques Used: Western Blot

    Dnmt1 is ubiquitinated in vivo and interacts with Cdh1, the substrate recognition subunit of APC/C Cdh1 (E3 ligase) which is involved in its degradation and which is dephosphorylated upon 5-aza-CdR treatment. (A) Dnmt1 is ubiquitinated in vivo. Cos-7 cells
    Figure Legend Snippet: Dnmt1 is ubiquitinated in vivo and interacts with Cdh1, the substrate recognition subunit of APC/C Cdh1 (E3 ligase) which is involved in its degradation and which is dephosphorylated upon 5-aza-CdR treatment. (A) Dnmt1 is ubiquitinated in vivo. Cos-7 cells

    Techniques Used: In Vivo

    7) Product Images from "6-Thioguanine Reactivates Epigenetically Silenced Genes in Acute Lymphoblastic Leukemia Cells by Facilitating Proteasome-mediated Degradation of DNMT1"

    Article Title: 6-Thioguanine Reactivates Epigenetically Silenced Genes in Acute Lymphoblastic Leukemia Cells by Facilitating Proteasome-mediated Degradation of DNMT1

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-3430

    DNMT1 was degraded upon S G treatment and the degradation could be blocked by a proteasomal inhibitor
    Figure Legend Snippet: DNMT1 was degraded upon S G treatment and the degradation could be blocked by a proteasomal inhibitor

    Techniques Used:

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

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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, 2022-08
    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: In brief, homology modeling of the human DNMT1 (hDNMT1) utilized the crystal structure of mouse DNMT1 (mDNMT1) in complex with hemi-methylated DNA (PDB 4DA4) was carried out based on the human reference sequence ( ). mDNMT1 shares an 85% sequence similarity with the hDNMT1 reference sequence ( ).

    Techniques: Binding Assay, Methylation

    Western blot results for DNMT1 (A), LSD1 (B), and Set7 (C) and DNMT1 with monomethylation on K142 (D) in the whole cell lysate of Jurkat-T cells treated with 10 µM cyclophosphamide for the indicated periods of time. The culture medium was changed

    Journal: Chemical research in toxicology

    Article Title: Cyclophosphamide Perturbs Cytosine Methylation in Jurkat-T Cells through LSD1-mediated Stabilization of DNMT1 Protein

    doi: 10.1021/tx2003849

    Figure Lengend Snippet: Western blot results for DNMT1 (A), LSD1 (B), and Set7 (C) and DNMT1 with monomethylation on K142 (D) in the whole cell lysate of Jurkat-T cells treated with 10 µM cyclophosphamide for the indicated periods of time. The culture medium was changed

    Article Snippet: Transferring to Protran nitrocellulose membrane (Whatman, Piscataway, NJ) was carried out by electroblotting in transfer buffer (10 mM NaHCO3 , 3 mM Na2 CO3 , pH 9.9, 20% methanol and 0.1% SDS) at 30 V for 12 h. Antibodies that specifically recognize human DNMT1 (New England Biolabs, Ipswich, MA), DNMT1-K142me , DNMT3a (Abcam, San Francisco, CA), DNMT3b (Abcam), human LSD1 (Cell Signaling, Danvers, MA), Set7 (Cell Signaling) and human β-actin (Abcam, Cambridge, MA) were used at 1:5000, 1:1000, 1:10000, 1:20000, 1:5000, 1:5000, and 1:10000 dilution, respectively.

    Techniques: Western Blot

    ChIP analysis of DMR1 occupancy by Ctcf, Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific DNA sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P

    Journal: Biochemical Journal

    Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

    doi: 10.1042/BJ20111417

    Figure Lengend Snippet: ChIP analysis of DMR1 occupancy by Ctcf, Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific DNA sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P

    Article Snippet: Then the resin was collected and incubated with 0.4 pmol of human recombinant PARP1 (Alexis) and/or 0.2 pmol of human recombinant DNMT1 (New England Biolabs) in 300 μl of native purification buffer for 3 h on a rotative shaker at 4°C.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification

    Analysis of Ctcf–Parp1–Dnmt1 interactions and DMR1 region occupancy following PARG overexpression ( A ) Western blot analysis of nuclear lysates from L929 cells overexpressing PARG after 24 and 72 h of puromycin selection. Analyses were performed by anti-PARs, anti-Myc Abs for exogenous PARG and anti-Sp1 Ab as a control. ( B ) Immunoprecipitation of Ctcf in nuclear lysates from L929 cells overexpressing PARG 48 h after transfection. ( C ) ChIP analysis was carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs in L929 cells overexpressing PARG compared with controls after 72 h of puromycin selection. Real-time PCR data are expressed as the percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. Results are means±S.E.M. for three experiments. The Actb promoter was used as negative control. * P

    Journal: Biochemical Journal

    Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

    doi: 10.1042/BJ20111417

    Figure Lengend Snippet: Analysis of Ctcf–Parp1–Dnmt1 interactions and DMR1 region occupancy following PARG overexpression ( A ) Western blot analysis of nuclear lysates from L929 cells overexpressing PARG after 24 and 72 h of puromycin selection. Analyses were performed by anti-PARs, anti-Myc Abs for exogenous PARG and anti-Sp1 Ab as a control. ( B ) Immunoprecipitation of Ctcf in nuclear lysates from L929 cells overexpressing PARG 48 h after transfection. ( C ) ChIP analysis was carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs in L929 cells overexpressing PARG compared with controls after 72 h of puromycin selection. Real-time PCR data are expressed as the percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. Results are means±S.E.M. for three experiments. The Actb promoter was used as negative control. * P

    Article Snippet: Then the resin was collected and incubated with 0.4 pmol of human recombinant PARP1 (Alexis) and/or 0.2 pmol of human recombinant DNMT1 (New England Biolabs) in 300 μl of native purification buffer for 3 h on a rotative shaker at 4°C.

    Techniques: Over Expression, Western Blot, Selection, Immunoprecipitation, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Ctcf, Parp1, and Dnmt1 bind to non-methylated DNA molecules within the DMR1 Ctcf, Parp1, and Dnmt1 ChIPs were performed for the DMR1 b and c fragments. End-point PCR was performed after digestion of ChIP fractions DNA and inputs with either HpaII (H, methylation sensitive) or MspI (M, methylation insensitive) following heat inactivation of the restriction enzymes. The input (4% of the chromatin subjected to immunoprecipitation) was diluted 1/100 or 1/200 before restriction. The uncut (U) fractions consisted in HpaII digestions preventively blocked by heat inactivation. W, PCR performed in the absence of added template.

    Journal: Biochemical Journal

    Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

    doi: 10.1042/BJ20111417

    Figure Lengend Snippet: Ctcf, Parp1, and Dnmt1 bind to non-methylated DNA molecules within the DMR1 Ctcf, Parp1, and Dnmt1 ChIPs were performed for the DMR1 b and c fragments. End-point PCR was performed after digestion of ChIP fractions DNA and inputs with either HpaII (H, methylation sensitive) or MspI (M, methylation insensitive) following heat inactivation of the restriction enzymes. The input (4% of the chromatin subjected to immunoprecipitation) was diluted 1/100 or 1/200 before restriction. The uncut (U) fractions consisted in HpaII digestions preventively blocked by heat inactivation. W, PCR performed in the absence of added template.

    Article Snippet: Then the resin was collected and incubated with 0.4 pmol of human recombinant PARP1 (Alexis) and/or 0.2 pmol of human recombinant DNMT1 (New England Biolabs) in 300 μl of native purification buffer for 3 h on a rotative shaker at 4°C.

    Techniques: Methylation, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    Analysis of Ctcf interaction with Parp1 and Dnmt1 ( a ) Reciprocal co-IP of Ctcf, Parp1 and Dnmt1 in nuclear lysates. Non-specific rabbit IgGs were used as negative control. ( b ) Immunoprecipitation of Ctcf in nuclear lysates from L929 (Parp1 proficient) and A1 (Parp1 −/− ) cells. ( c ) Pull-down assay with recombinant brCtcf (His-tagged), Parp1 and Dnmt1 proteins.

    Journal: Biochemical Journal

    Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

    doi: 10.1042/BJ20111417

    Figure Lengend Snippet: Analysis of Ctcf interaction with Parp1 and Dnmt1 ( a ) Reciprocal co-IP of Ctcf, Parp1 and Dnmt1 in nuclear lysates. Non-specific rabbit IgGs were used as negative control. ( b ) Immunoprecipitation of Ctcf in nuclear lysates from L929 (Parp1 proficient) and A1 (Parp1 −/− ) cells. ( c ) Pull-down assay with recombinant brCtcf (His-tagged), Parp1 and Dnmt1 proteins.

    Article Snippet: Then the resin was collected and incubated with 0.4 pmol of human recombinant PARP1 (Alexis) and/or 0.2 pmol of human recombinant DNMT1 (New England Biolabs) in 300 μl of native purification buffer for 3 h on a rotative shaker at 4°C.

    Techniques: Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, Pull Down Assay, Recombinant