pet28 mhl  (Addgene inc)

Journal: Blood Advances

Article Title: Double mutant DNMT3A AML: a unique subtype experiencing increased DNA damage and poor prognosis ∗ †

doi: 10.1182/bloodadvances.2024014698

Figure Lengend Snippet: WT DNMT3A potentiates the glycosylase activity of TDG but not MBD4. (A) Schematic overview of the DNA (de)methylation cascade in humans, including de novo methylation, spontaneous deamination of 5mC, and BER of G-T mismatches. (B) Experimental setup of glycosylase assay. A FAM-labeled double-stranded 32-bp oligo, containing a G-T mismatch, is incubated with TDG (AA 111-348). Functional TDG identifies and excises the mismatch. The subsequent hot alkaline treatment facilitates the oligo to break at the site of the excised base, resulting in an 18-bp product. More functional TDG results in increased levels of product detected on gel. (C-D) Addition of WT DNMT3A to the glycosylase assay stimulates TDG activity in a dose-dependent matter; represented on gel (C) and quantified relative to unstimulated TDG (D). (E-F) Glycosylase activity of MBD4 is not stimulated by the addition of WT DNMT3A to the glycosylase assay; represented on gel (E) and quantified relative to unstimulated MBD4 (F). 5caC, 5-carboxylcytosine; 5fC, 5-formylcytosine; 5hmC, 5-hydroxymethylcytosine; FAM, fluorescein amidite.

Article Snippet: The pET28-MHL hexahistidine-tagged DNMT3A WT and mutants, MBD4 residues 430-580 (pET28, Addgene, Watertown, MA), and TDG residues 111 to 348 (pET28; kindly provided by Hashimoto et al) were expressed in Escherichia coli BL21(DE3) Gold and BL21(DE3) pLysS cells (Life Technologies, Carlsbad, CA).

Techniques: Activity Assay, Methylation, Labeling, Incubation, Functional Assay

Journal: Blood Advances

Article Title: Double mutant DNMT3A AML: a unique subtype experiencing increased DNA damage and poor prognosis ∗ †

doi: 10.1182/bloodadvances.2024014698

Figure Lengend Snippet: DNMT3A mutants commonly found in MBD4-deficient patients impair the glycosylase activity of TDG. (A) Gel image of glycosylase assay with mutant DNMT3A. Addition of mutant DNMT3A does not potentiate TDG activity and even inhibits TDG activity at higher concentrations. (B-E) Quantification of each DNMT3A mutant tested in panel A, relative to unstimulated TDG.

Article Snippet: The pET28-MHL hexahistidine-tagged DNMT3A WT and mutants, MBD4 residues 430-580 (pET28, Addgene, Watertown, MA), and TDG residues 111 to 348 (pET28; kindly provided by Hashimoto et al) were expressed in Escherichia coli BL21(DE3) Gold and BL21(DE3) pLysS cells (Life Technologies, Carlsbad, CA).

Techniques: Activity Assay, Mutagenesis

Journal: Blood Advances

Article Title: Double mutant DNMT3A AML: a unique subtype experiencing increased DNA damage and poor prognosis ∗ †

doi: 10.1182/bloodadvances.2024014698

Figure Lengend Snippet: Cotitration of increasing amounts of recombinant WT DNMT3A relative to different mutant counterparts. (A-D) Cotitration of WT DNMT3A compared to DNMT3A R635W (A), DNMT3A R688C (B), DNMT3A R882C (C) and DNMT3A A884V (D). ∗Samples run on different gels in the same experiment and imaged at the same time due to logistic limitations.

Article Snippet: The pET28-MHL hexahistidine-tagged DNMT3A WT and mutants, MBD4 residues 430-580 (pET28, Addgene, Watertown, MA), and TDG residues 111 to 348 (pET28; kindly provided by Hashimoto et al) were expressed in Escherichia coli BL21(DE3) Gold and BL21(DE3) pLysS cells (Life Technologies, Carlsbad, CA).

Techniques: Recombinant, Mutagenesis

Journal: Blood Advances

Article Title: Double mutant DNMT3A AML: a unique subtype experiencing increased DNA damage and poor prognosis ∗ †

doi: 10.1182/bloodadvances.2024014698

Figure Lengend Snippet: Patient and mutation characteristics by DNMT3A allelic state. (A) Classification of DNMT3A mutants. (B) Frequency of driver mutations per DNMT3A mutant group (SM [left] and DM [right]). The percentage in which each individual gene occurs is shown behind each bar. (C) OS of DNMT3A SM (blue line) and DNMT3A DM (green line). (D) Lollipop plot illustrating the distribution of mutations in the DNMT3A SM cohort (upper part) and DNMT3A DM cohort (bottom part). The length of a lollipop represents the number of patients that carry a mutation at a specific amino acid. Each point is colored by the mutation type of the most frequent occurring mutation at that specific location. (E) Combination of type of mutations in DNMT3A DMs with heterozygous mutations. SNV, single nucleotide variant.

Article Snippet: The pET28-MHL hexahistidine-tagged DNMT3A WT and mutants, MBD4 residues 430-580 (pET28, Addgene, Watertown, MA), and TDG residues 111 to 348 (pET28; kindly provided by Hashimoto et al) were expressed in Escherichia coli BL21(DE3) Gold and BL21(DE3) pLysS cells (Life Technologies, Carlsbad, CA).

Techniques: Mutagenesis, Variant Assay

Journal: Blood Advances

Article Title: Double mutant DNMT3A AML: a unique subtype experiencing increased DNA damage and poor prognosis ∗ †

doi: 10.1182/bloodadvances.2024014698

Figure Lengend Snippet: Characteristics of patients selected for WGS

Article Snippet: The pET28-MHL hexahistidine-tagged DNMT3A WT and mutants, MBD4 residues 430-580 (pET28, Addgene, Watertown, MA), and TDG residues 111 to 348 (pET28; kindly provided by Hashimoto et al) were expressed in Escherichia coli BL21(DE3) Gold and BL21(DE3) pLysS cells (Life Technologies, Carlsbad, CA).

Techniques: Mutagenesis

Journal: Blood Advances

Article Title: Double mutant DNMT3A AML: a unique subtype experiencing increased DNA damage and poor prognosis ∗ †

doi: 10.1182/bloodadvances.2024014698

Figure Lengend Snippet: Methylation damage burden is increased in patients carrying 2 DNMT3A mutations, particularly when these mutations reside at the DNMT3A-TDG interaction surface. (A) Box plot for SBS1 score grouped by DNMT3A mutant group. Each point depicts the SBS1 score of an individual patient. The color of each data point represents the quantity of DNMT3A mutations situated within the DNMT3A-TDG interface. (B) Box plots for SBS1 score grouped by the number of DNMT3A mutations located at the DNMT3A-TDG interaction surface. The color of each data point represents the DNMT3A mutant group.

Article Snippet: The pET28-MHL hexahistidine-tagged DNMT3A WT and mutants, MBD4 residues 430-580 (pET28, Addgene, Watertown, MA), and TDG residues 111 to 348 (pET28; kindly provided by Hashimoto et al) were expressed in Escherichia coli BL21(DE3) Gold and BL21(DE3) pLysS cells (Life Technologies, Carlsbad, CA).

Techniques: Methylation, Mutagenesis