tdg protein nuclear expression  (Human Protein Atlas)

Journal: Journal of Central South University Medical Sciences

Article Title: Levels of base excision repair proteins in CD4 + T cells in patients with systemic lupus erythematosus

doi: 10.11817/j.issn.1672-7347.2022.210485

Figure Lengend Snippet: A, C: Comparison of the AID protein expression levels in CD4 + T cells between SLE patients and healthy controls; A, D: Comparison of the TDG protein expression levels in CD4 + T cells between SLE patients and healthy controls; B, E: Comparison of the MBD4 protein expression levels in CD4 + T cells between SLE patients and healthy controls. SLE: Systemic lupus erythematosus.

Article Snippet: In the Human Protein Atlas database, the TDG protein nuclear expression in melanoma maintains a medium-to-high expression and high level is related to adverse outcomes.

Techniques: Comparison, Expressing

Journal: Oncogene

Article Title: Thymine DNA glycosylase as a novel target for melanoma

doi: 10.1038/s41388-018-0640-2

Figure Lengend Snippet: TDG inhibitors decrease clonogenic capacity and cell viability in melanoma cells. a , b Molecular beacon DNA repair assay showing dose-dependent inhibition of TDG glycosylase activity by juglone and closantel, respectively; fluorescence is in arbitrary units (AU); time is in 0.5-s intervals. c , d Conventional TDG glycosylase assays, in which G:T mismatch-bearing double-stranded oligonucleotide substrates and 32 P-labeled at the 3′ end on the mismatched T-containing strand were treated with purified recombinant TDG protein at 37 °C; the reactions were then treated with 100 mM NaOH at 90 °C for 30’ in order to cleave the sugar-phosphate backbone at the AP site and the resulting reaction product was separated from the longer substrate by PAGE. Juglone and closantel were tested at concentrations of 100 nM, 10 μM, and 1 mM. A negative control is included. e , f Cell viability of SK28 melanoma cells treated with increasing doses of juglone and closantel. g , h Clonogenic capacity of SK28 melanoma cells treated with different doses of juglone and closantel or DMSO as a control; i , j corresponding stained plates. All the experiments were performed in duplicate or triplicate and data, normalized to DMSO, are presented as average ± standard deviation. k , l Detection by immunofluorescence of 5caC in juglone- and closantel-treated SK28 cells; DMSO was used as a negative control; nuclei were counter-stained with DAPI. m , n Fluorescence-activated cell sorting (FACS) of propidium iodide-stained Mel501 and SK28 cells, showing that juglone induces cell cycle arrest in G2–M phase and increased S phase/>4 n cells, respectively

Article Snippet: In the Human Protein Atlas database, nuclear expression of TDG protein is also maintained at high-to-medium levels in melanomas; and high expression is associated with unfavorable prognosis (Suppl.

Techniques: Inhibition, Activity Assay, Fluorescence, Labeling, Purification, Recombinant, Negative Control, Control, Staining, Standard Deviation, Immunofluorescence, FACS

Journal: DNA repair

Article Title: Activity and crystal structure of human thymine DNA glycosylase mutant N140A with 5-carboxylcytosine DNA at low pH

doi: 10.1016/j.dnarep.2013.04.003

Figure Lengend Snippet: Data collection and refinement statistics.

Article Snippet: TDG N140A mutant protein (0.35 mM) was mixed with 0.2 mM of annealed 28-bp oligonucleotide (synthesized by the New England Biolabs, Inc.): 5′-CAG CTC TGT ACG TGA GCG ATG GAC AGC T-3′ and 5′-AGC TGT CCA TCG CTC AXG TAC AGA GCT G-3′ where X is 5caC.

Techniques: Mutagenesis

Journal: DNA repair

Article Title: Activity and crystal structure of human thymine DNA glycosylase mutant N140A with 5-carboxylcytosine DNA at low pH

doi: 10.1016/j.dnarep.2013.04.003

Figure Lengend Snippet: Mutations of three asparagine residues in the active site of TDG; the activities of TDG wild type (WT) (a), mutants N140A (b), N140D (c), N230D (d), N157A (e), N157D (f), N157D/N230D (g), and N140D/N157D (h). (i) Summary of relative activities (by fold reduction from that of WT) of mutants for G:U and G:5caC substrates (NA: no activity observed).

Article Snippet: TDG N140A mutant protein (0.35 mM) was mixed with 0.2 mM of annealed 28-bp oligonucleotide (synthesized by the New England Biolabs, Inc.): 5′-CAG CTC TGT ACG TGA GCG ATG GAC AGC T-3′ and 5′-AGC TGT CCA TCG CTC AXG TAC AGA GCT G-3′ where X is 5caC.

Techniques: Activity Assay

Journal: DNA repair

Article Title: Activity and crystal structure of human thymine DNA glycosylase mutant N140A with 5-carboxylcytosine DNA at low pH

doi: 10.1016/j.dnarep.2013.04.003

Figure Lengend Snippet: Structure of TDG N140A mutant in complex with G:5caC containing DNA: (a) 2Fo-Fc electron density, contoured at 1σ above the mean, for the entire 28-bp DNA(blue) and human TDG glycosylase domain (green) used in the structure determination; (b) an enlarged active site with the 5caC base in the middle and TDG active site residues in green. The omit electron densities, after omitting the 5caC base, are shown as 2Fo-Fc in 1σ (gray) and Fo-Fc in 5σ (blue). The Fo-Fc in 3σ shows a similar 5caC envelope as that of 2Fo-Fc (not shown). The modeled 5caC base conformation represents an averaged conformation of TDG-DNA substrate and product complexes in the crystal, with the distance between the C1′ and N1 atoms at approximately 2.3 Å, obviously longer than the N-glycosidic bond distance of 1.5 Å; (c) superimposition of a post-reactive complex with an a basic sugar with a hydrolyzed C1′ (colored in cyan; PDB 4FNC) [19]; (d) superimposition of a pre-reactive complex (colored in salmon pink; PDB 3UFJ) [18] shows a putative nucleophilic water molecule (in black), held in position by the side chain carbonyl oxygen atom of Asn140, attacking the C1′ from the opposite side of the leaving base. The numerical numbers indicate relative movement (in Angstroms) of residue Thr197 and the water molecule. The omit Fo-Fc electron density is contoured in 5σ (blue); (e)two possible catalytic mechanisms for TDG: a concerted, synchronous SN2 mechanism (top) and a dissociated (asynchronous) SN1 mechanism proceeding through a discrete transition state (bottom). The reactant (left) and the product (right) are shown. The structural feature for the reactant is adopted from a pre-reactive complex of WT TDG with a uracil analog (2′-deoxy-2′-fluoroarabinouridine) (PDB 3UFJ) [18]. The structural feature for the product is adopted from a post-reactive complex of WT TDG with a G:5hmU mismatch (PDB 4FNC) that generated a C1′ hydrolyzed a basic sugar [19]. E. coli UDG follows a discrete oxocarbenium ion intermediate (bottom) [33]. The question mark signals a different reaction mechanism employed by TDG; (f) hydrophobic interactions between the 5caC base and Gly138 and Ile139; (g) the leaving 5caC base interacts with side chains of Ser271, Asn230 and Tyr152. The numerical numbers indicate hydrogen bond distances in Angstroms; (h) superimposition of N140A in complex with a 5caC-containing 22-bp DNA crystallized in pH 7 (colored in yellow; PDB 3UO7) [20] and 5caC-containing 28-bp DNA crystallized in pH 4.6 (colored in green and gray; this study) suggests a base rotation of approximately 120° around the glycoside bond.

Article Snippet: TDG N140A mutant protein (0.35 mM) was mixed with 0.2 mM of annealed 28-bp oligonucleotide (synthesized by the New England Biolabs, Inc.): 5′-CAG CTC TGT ACG TGA GCG ATG GAC AGC T-3′ and 5′-AGC TGT CCA TCG CTC AXG TAC AGA GCT G-3′ where X is 5caC.

Techniques: Mutagenesis, Generated