recombinant dnmt1  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Phosphorus-containing analogues of methyltransferase substrates/inhibitors and selected reactions catalyzed by these enzymes. ( a ) Structures of the H-phosphinic analogue of methionine (L-Met-P H ), (S,S)-SAM, H -phosphinic analogues of SAM and SAH ((R,S)-SAM-P H and rac-SAH-P H ), and phosphonic analogues of SAM and SAH (rac-SAM-P 5 and rac-SAH-P 5 ). H-Phosphinic and phosphonic groups are marked in yellow and rose, respectively. Individual diastereomers have hashed wedge bonds at the chiral centres, while the corresponding racemates are depicted using solid bonds. ( b ) DNA-(cytosine C5) methylation at CpG sites catalyzed by Dnmt3a, which establishes de novo DNA methylation patterns , and methylation at hemimethylated CpG sites catalyzed by Dnmt1, which maintains DNA methylation patterns during replication in mammals . ( c ) Methylation of protocatechuic aldehyde catalyzed by catechol-O-methyltransferase (COMT).

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Analogues, Methylation, DNA Methylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Effect of SAM phosphorus-containing analogues on Dnmt1 activity. ( a ) Methylation of D1 hairpin DNA fragment by Dnmt1 in a time-dependent and SAM concentration-dependent manner at 37 °C for 4–24 h using 1.0 µM and 5.0 µM SAM. ( b ) Dnmt1 methylation activity (24 h) in the presence of SAM phosphorus-containing analogues: rac-SAM-P H (100 µM), (R,S)-SAM-P H (20 µM and 50 µM), or rac-SAM-P 5 (20 µM and 100 µM), compared with SAM (5 µM). In both panels, the control was D2 hairpin DNA (a product of methylation) digested with GlaI. In both panels the results indicate means from five independent experiments. Error bars represent standard deviations. ** p ≤ 0.01 according to one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Analogues, Activity Assay, Methylation, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Interaction of SAM and SAH phosphorus-containing analogues with Dnmt1 and COMT. ( a ) Inhibition of Dnmt1 methylation activity. Dnmt1 reaction mixtures contained the following: SAM (5 µM), rac-SAH-P 5 (100 µM), rac-SAM-P 5 (100 µM), rac-SAH-P H (100 µM), or (R,S)-SAM-P H (100 µM). SAH (25 µM) was used as a reference inhibitor. The D2 DNA hairpin, the product of the Dnmt1 reaction cleaved with GlaI, was used as the control. ( b ) Inhibition of COMT methylation activity. Reaction mixtures contained either SAM or (R,S)-SAM-P H (each 6 mM) as methyl group donors and SAH (3 mM) or rac-SAH-P H (3 mM) as inhibitors. The duration of the reactions was 2 h and 24 h. In both panels the results indicate means from five independent experiments. Error bars represent standard deviations. ** p ≤ 0.01 according to one-way ANOVA with Dunnett’s post hoc test (vs. “no inhibitor”).

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Analogues, Inhibition, Methylation, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Phospho-Switch: Regulation of the Activity of SAM-Dependent Methyltransferases Using H -Phosphinic SAM Analogue

doi: 10.3390/ijms26178590

Figure Lengend Snippet: Dnmt1 activity assay using a hemimethylated DNA hairpin D1 and the endonuclease GlaI, making a double-strand break into the fully methylated hairpin D2 that results in enhanced fluorescence. The methylated CpG site is located within the endonuclease GlaI recognition site (GCGm 5 C/Gm 5 CGm 5 C).

Article Snippet: Human recombinant DNA methyltransferase Dnmt1 was purchased from ThermoFisher (Waltham, MA, USA).

Techniques: Activity Assay, Methylation, Fluorescence

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: Interactions of BACH2 IDR with TBK1 and DNMT1 (A) Upper: a schematic representation of mouse BACH2 and BACH2-IDR. Lower: the immunoblot analysis of the affinity-purified samples using an anti-FLAG, anti-TBK1 or anti-DNMT1 antibody. (B) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, or His-FLAG-TBK1 in the presence or absence of 5 μM or10 μM heme. GST and GST-BACH2-IDR were revealed with an anti-GST antibody. TBK1 was revealed with an anti-TBK1 antibody. (C) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, or His-FLAG-DNMT1 in the presence or absence of 5 μM or 10 μM heme. GST and GST-BACH2-IDR were revealed with an anti-GST antibody. DNMT1 was revealed with an anti-DNMT1antibody. (D) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, and B cell extracts in the presence or absence of 10 μM heme. GST and GST-BACH2-IDR were revealed with anti-GST an antibody. TBK1 was revealed with an anti-TBK1 antibody. DNMT1 was revealed with an anti-DNMT1 antibody.

Article Snippet: His-FLAG-DNMT1 , BPS Bioscience , Cat#51100.

Techniques: Western Blot, Affinity Purification, In Vitro, Pull Down Assay, Recombinant

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: The list of proteins identified as BACH2_IDR interacting proteins using ReCLIP

Article Snippet: His-FLAG-DNMT1 , BPS Bioscience , Cat#51100.

Techniques: Sequencing

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet:

Article Snippet: His-FLAG-DNMT1 , BPS Bioscience , Cat#51100.

Techniques: Virus, Recombinant, Protease Inhibitor, Transfection, Protein Purification, Reporter Assay, Luciferase, Cell Isolation, Plasmid Preparation, Reverse Transcription, SYBR Green Assay, Sequencing, Negative Control, Software

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: Interactions of BACH2 IDR with TBK1 and DNMT1 (A) Upper: a schematic representation of mouse BACH2 and BACH2-IDR. Lower: the immunoblot analysis of the affinity-purified samples using an anti-FLAG, anti-TBK1 or anti-DNMT1 antibody. (B) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, or His-FLAG-TBK1 in the presence or absence of 5 μM or10 μM heme. GST and GST-BACH2-IDR were revealed with an anti-GST antibody. TBK1 was revealed with an anti-TBK1 antibody. (C) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, or His-FLAG-DNMT1 in the presence or absence of 5 μM or 10 μM heme. GST and GST-BACH2-IDR were revealed with an anti-GST antibody. DNMT1 was revealed with an anti-DNMT1antibody. (D) The in vitro pull-down assay using recombinant GST, GST-BACH2-IDR, and B cell extracts in the presence or absence of 10 μM heme. GST and GST-BACH2-IDR were revealed with anti-GST an antibody. TBK1 was revealed with an anti-TBK1 antibody. DNMT1 was revealed with an anti-DNMT1 antibody.

Article Snippet: Recombinant human His-FLAG DNMT1 was purchased from BPS Bioscience.

Techniques: Western Blot, Affinity Purification, In Vitro, Pull Down Assay, Recombinant

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: The list of proteins identified as BACH2_IDR interacting proteins using ReCLIP

Article Snippet: Recombinant human His-FLAG DNMT1 was purchased from BPS Bioscience.

Techniques: Sequencing

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet:

Article Snippet: Recombinant human His-FLAG DNMT1 was purchased from BPS Bioscience.

Techniques: Virus, Recombinant, Protease Inhibitor, Transfection, Protein Purification, Reporter Assay, Luciferase, Cell Isolation, Plasmid Preparation, Reverse Transcription, SYBR Green Assay, Sequencing, Negative Control, Software

Journal: PLOS ONE

Article Title: Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy

doi: 10.1371/journal.pone.0307850

Figure Lengend Snippet: (A) Domain organization of human DNMT1. (B) Representative 2D class averages calculated from micrographs showing different particle orientations. (C) Cryo-EM density map of apo DNMT1 in front and side views. Domains are color-coded as in (A). (D) Cryo-EM density map colored according to the resolution distribution at higher (upper panel) and lower (lower panel) contour levels. The resolution (Å) is color-coded as indicated on the bar. (E) Representative regions of the cryo-EM map with the crystal structure of apo human DNMT1 (PDB 4WXX) fitted in.

Article Snippet: Recombinant human full-length DNMT1 was expressed by Genscript and purified by GSK with the following steps: Expression of DNMT1 with N-terminal tag containing Flag-6xHis-TEV protease cleavage site was performed using the baculovirus/ Sf 9 insect cell expression system.

Techniques: Cryo-EM Sample Prep

Journal: PLOS ONE

Article Title: Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy

doi: 10.1371/journal.pone.0307850

Figure Lengend Snippet: Cryo-EM data collection of apo DNMT1, DNMT1: Non-productive DNA complex, DNMT1: H3Ub2-peptide, DNMT1: Productive DNA complex with refinement statistics for apo DNMT1.

Article Snippet: Recombinant human full-length DNMT1 was expressed by Genscript and purified by GSK with the following steps: Expression of DNMT1 with N-terminal tag containing Flag-6xHis-TEV protease cleavage site was performed using the baculovirus/ Sf 9 insect cell expression system.

Techniques: Biomarker Discovery

Journal: PLOS ONE

Article Title: Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy

doi: 10.1371/journal.pone.0307850

Figure Lengend Snippet: (A) Binding of DNMT1 to DNA in the presence and absence of GSK3852279B inhibitor (labeled as [C]) analysed by native gel electrophoresis. The gel is stained to detect either protein (left) or DNA (right). (B) Cryo-EM density map and 2D analysis of the DNMT1: DNA: GSK3852279B complex showing the non-productive state. The DNMT1 domains are colored as in . The DNA density is indicated by yellow arrows (2D) and colored in orange (3D). Crystal structure PDB 3PTA has been placed into the cryo-EM density. (C) DNA methylation by DNMT1 in the presence of H3Ub2-peptide and GSK3852279B inhibitor (labeled as [C]) analyzed by SDS-PAGE. The gel is stained to detect either protein (left) or DNA (right). Free DNMT1 and DNMT1 cross-linked to 5FDNA (DNMT1-5FDNA) are indicated. (D) Cryo-EM density map and 2D analysis of the DNMT1:5FDNA complex in the presence of H3Ub2-peptide showing the productive state. The DNMT1 domains are colored as in . The DNA density is indicated by yellow arrows (2D) and colored in orange (3D). Crystal structure PDB 4DA4 has been placed into the cryo-EM density.

Article Snippet: Recombinant human full-length DNMT1 was expressed by Genscript and purified by GSK with the following steps: Expression of DNMT1 with N-terminal tag containing Flag-6xHis-TEV protease cleavage site was performed using the baculovirus/ Sf 9 insect cell expression system.

Techniques: Binding Assay, Labeling, Nucleic Acid Electrophoresis, Staining, Cryo-EM Sample Prep, DNA Methylation Assay, SDS Page

Journal: PLOS ONE

Article Title: Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy

doi: 10.1371/journal.pone.0307850

Figure Lengend Snippet: (A) Cryo-EM density map showing DNMT1 (grey) and additional densities for the two ubiquitin molecules of the histone H3 peptide analogue (wheat) in front and side views. The sequence of the H3 peptide and the positions of Ub-18 and Ub-23 attached to it (red boxes) are indicated in the lower panel. (B) Superposition of the RFTS domain from apo DNMT1 (PDB ID 4WXX; RFTS in dark blue; other DNMT1 domains shown in grey surface) and RFTS from the RFTS-H3Ub2 crystal structure (PDB ID 5WVO; RFTS in light blue; ubiquitin molecules in grey cartoon). The bend in the α4-helix of RFTS is indicated by a red arrow and shown in the close-up view on the right. (C) Two alternative fits (on the left and right) of the RFTS: H3Ub2 crystal structure (PDB ID 5WVO, light blue RFTS, grey H3Ub2) into the cryo-EM density map of DNMT1: H3Ub2-peptide (lower panel). RFTS domain from the apo DNMT1 crystal structure (dark blue, upper panel) is provided as reference. (D) Fit of the apo DNMT1 structure (PDB ID 4WXX, domains colored as in ) and the two ubiquitin molecules from the RFTS: H3Ub2 structure (PDB ID 5WVO) into the cryo-EM map of DNMT1: H3Ub2-peptide.

Article Snippet: Recombinant human full-length DNMT1 was expressed by Genscript and purified by GSK with the following steps: Expression of DNMT1 with N-terminal tag containing Flag-6xHis-TEV protease cleavage site was performed using the baculovirus/ Sf 9 insect cell expression system.

Techniques: Cryo-EM Sample Prep, Ubiquitin Proteomics, Sequencing

Journal: PLOS ONE

Article Title: Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy

doi: 10.1371/journal.pone.0307850

Figure Lengend Snippet: Apo DNMT1 is in an auto-inhibited state with the RFTS domain blocking access of the DNA to the catalytic domain (left panel). Binding of hemi-methylated DNA in a non-productive state and/or di-ubiquitinated histone H3 releases the autoinhibition of apo DNMT1 (middle panel). DNMT1 transitions into a productive state where hemi-methylated DNA can enter the active site to become fully methylated (right panel).

Article Snippet: Recombinant human full-length DNMT1 was expressed by Genscript and purified by GSK with the following steps: Expression of DNMT1 with N-terminal tag containing Flag-6xHis-TEV protease cleavage site was performed using the baculovirus/ Sf 9 insect cell expression system.

Techniques: Blocking Assay, Binding Assay, Methylation