Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Quantitative analysis of hSMUG1-mediated uracil excision using nucleotide MALDI-TOF mass spectrometry. (A) Schematic representation of experimental design. A duplex DNA substrate was generated by annealing an 18-nucleotide lesion strand containing a signal uridine (U) with a complementary 19-nucleotide template strand (T), thereby introducing a U:G mispair at the centre of the duplex. Recombinant hSMUG1 catalysed the monofunctional glycosylase reaction, excising uracil to yield an abasic (AP site on the lesion strand called abasic primer (AP). The reaction products were subsequently profiled by nucleotide MALDI-TOF MS. Distinct m/z peaks corresponding to the U-, T-, and AP-containing strands enabled direct quantification of the excision reaction. Notably, uracil removal resulted in a 94 Da mass decrease, allowing precise discrimination between U and AP fragments. (B) In the absence of hSMUG1, the duplex exhibited two distinct peaks at 5789.8 Da (T strand) and 5541.6 Da (U strand) with high signal-to-noise ratios, confirming substrate integrity and mass resolution. (C) Upon hSMUG1 treatment, the uracil-containing (U) peak was predominantly converted into the AP peak at 5447.6 Da, indicating efficient excision of uracil from the U:G substate. Excision efficiency was determined from peak intensities using the formula: AP/(AP+U) × U x 100%.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Mass Spectrometry, Generated, Recombinant

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Evaluation of hSMUG1 concentration-dependent uracil excision activity. A 10 μL hSMUG1 glycosylase reaction mixture contained 50 pmol of uracil-containing duplex substrate (5 μM), 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding various concentrations of hSMUG1 (0.5–2 U, as indicated). (A) Representative MALDI-TOF mass spectra obtained from reactions at different enzyme concentrations and incubation times. Distinct peaks corresponding to the template strand (T), uracil-containing strand (U), and abasic primer (AP) enabled direct visualisation of uracil excision efficiency. (B) Quantitative analysis of AP primer formation. Each reaction was independently performed in triplicate for each concentration and time point. Data are presented as mean ± SD. The dashed line represents the best-fit linear regression curve describing the relationship between hSMUG1 concentration and uracil excision efficiency.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Concentration Assay, Activity Assay, Incubation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Evaluation of hSMUG1-mediated uracil excision efficiency in damaged partners duplex or single-stranded DNA. A 10 μL hSMUG1 glycosylase reaction mixture contained 1 U hSMUG1, 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding various 50 pmol (5 μM) of uracil-containing substrates, including U:G or U:A mispair in the duplex DNA, or uracil in single-stranded DNA (ss U). (A) Representative MALDI-TOF mass spectra of reactions with different substrates and incubation times. Distinct peaks corresponding to the template strand (T or T U:A ), uracil-containing strand (U), and abasic product (AP) allowed direct visualisation of uracil excision efficiency. (B) Quantitative analysis of AP product formation. Each reaction was independently performed in triplicate for each substrate and time point. Data are presented as mean ± SD. Statistical analyses were performed by two-way ANOVA with Geisser-Greenhouse correction. **** p < 0.0001.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Incubation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: hSMUG1-mediated excision efficiency of U:G mispair at different positions within duplex DNA. A10 μL hSMUG1 glycosylase reaction mixture contained 1 U hSMUG1, 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding various 50 pmol (5 μM) of substrates containing U:G mispair positioned at the 3′ end, middle, or 5′ end of duplex DNA. (A) Representative MALDI-TOF mass spectra of reactions with different substrates and incubation times. Distinct peaks corresponding to the template strand (T or T 3′ or T 5′ ), uracil-containing strand (U or U 3′ or U 5′ ), and abasic primer (AP) allowed direct visualisation of uracil excision efficiency. (B) Quantitative analysis of AP product formation. Each reaction was independently performed in triplicate for each substrate and time point. Data are presented as mean ± SD. Statistical analyses were performed by two-way ANOVA with Geisser-Greenhouse correction. *** p < 0.001; **** p < 0.0001.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Incubation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Cadmium inhibits hSMUG1-mediated uracil excision: quantitative analysis and mitigation by Ganoderma lucidum extracts

doi: 10.1080/14756366.2026.2668792

Figure Lengend Snippet: Effect of cadmium on hSMUG1-mediated uracil excision from DNA. A10 μL hSMUG1 glycosylase reaction mixture contained 1 U hSMUG1, 10 mM MgCl 2 , 100 μg/mL, 100 μg/mL bovine serum albumin (BSA), and 10 mM Bis-Tris Propane-HCl buffer (pH 7.0, 25 °C). Reactions were initiated by adding 20 pmol substrate DNA in the presence of cadmium at various concentrations (0–50 μM). (A) Representative MALDI-TOF mass spectra of reactions with different cadmium concentrations and incubation times. Distinct peaks corresponding to the template strand (T), uracil-containing strand (U), and abasic primer (AP) allowed direct visualisation of uracil excision efficiency. (B) Inhibition curve of hSMUG1 activity by cadmium. Triplicate reactions performed under each cadmium concentration were used to generate an inhibition curve. The IC 50 of cadmium for hSMUG1 inhibition was 4.6 μM. The red arrow indicates the peak corresponding to the AP product. Data are presented as mean ± SD.

Article Snippet: Human single-strand selective Uracil-DNA Glycosylase (hSMUG1) and companion buffers were purchased from New England Biolabs, MA.

Techniques: Incubation, Inhibition, Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: Mitochondrial uracil DNA glycosylase contributes to nuclear base excision repair

doi: 10.64898/2026.04.30.721890

Figure Lengend Snippet: A) Schematic of the UNG locus in the human genome and the regions targeted for disruption with CRISPR/Cas9. B) Validation of UNG knockouts in 293T cells by immunoblotting and in vitro uracil excision assay [S, uracil-containing 43-mer ssDNA substrate; P, cleaved 30-mer ssDNA product; (-) = HED buffer only (negative control); (+) = 5 Units of E. coli uracil-DNA glycosylase (positive control for uracil excision)]. C) Fluorescence microscopy showing that UNG1/2 double-KO is necessary for full inhibition of nuclear UNG activity (scale=200μm; corresponding flow analysis in Supplementary Figure S4C ).

Article Snippet: Negative and positive controls with HED buffer and recombinant E. coli uracil-DNA glycosylase (New England Biolabs M0280S, lot 10272153), respectively, were included.

Techniques: Disruption, CRISPR, Biomarker Discovery, Western Blot, In Vitro, Excision Assay, Negative Control, Positive Control, Fluorescence, Microscopy, Inhibition, Activity Assay