udg  (New England Biolabs)


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  • 99
    Name:
    Uracil DNA Glycosylase UDG
    Description:
    Uracil DNA Glycosylase UDG 5 000 units
    Catalog Number:
    M0280L
    Price:
    296
    Size:
    5 000 units
    Category:
    DNA Glycosylases
    Score:
    85
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    Structured Review

    New England Biolabs udg
    Uracil DNA Glycosylase UDG
    Uracil DNA Glycosylase UDG 5 000 units
    https://www.bioz.com/result/udg/product/New England Biolabs
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    udg - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase"

    Article Title: Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

    Journal:

    doi: 10.1016/j.dnarep.2007.09.001

    hUgi inhibits UNG activity in 293 cells UNG-proficient cells (GFP) have a higher UDG activity than cells expressing hUgi (GFP-HUgi) for uracil in single-strand DNA (A) or double strand DNA (B). Increasing amounts of cell lysate for each cell line were incubated with 0.1 pmol of labeled oligonucleotide containing a single uracil. Cleavage results in generation of a truncated product. The negative control (−) reaction contained oligo in the absence of lysate. The lane labeled “UDG” was oligo incubated with purified UDG.
    Figure Legend Snippet: hUgi inhibits UNG activity in 293 cells UNG-proficient cells (GFP) have a higher UDG activity than cells expressing hUgi (GFP-HUgi) for uracil in single-strand DNA (A) or double strand DNA (B). Increasing amounts of cell lysate for each cell line were incubated with 0.1 pmol of labeled oligonucleotide containing a single uracil. Cleavage results in generation of a truncated product. The negative control (−) reaction contained oligo in the absence of lysate. The lane labeled “UDG” was oligo incubated with purified UDG.

    Techniques Used: Activity Assay, Expressing, Incubation, Labeling, Negative Control, Purification

    2) Product Images from "Functional Assessment of Population and Tumor-Associated APE1 Protein Variants"

    Article Title: Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065922

    Reconstitution assay using purified BER proteins. ( A ) Wild-type (WT) and variant APE1 proteins were incubated with UDG and POLβ with 32 P-labeled 34U DNA substrate (1 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing sequencing gel. The non-incised substrate (S), AP site incision product (P1), and gap-filling extension product (P2) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. ( B ) Relative AP site cleavage efficiency. Shown are the averages and standard deviations of 4 independent reactions. ( C ) Relative gap-filling activity. Shown are the average and standard deviation of 4 independent assays.
    Figure Legend Snippet: Reconstitution assay using purified BER proteins. ( A ) Wild-type (WT) and variant APE1 proteins were incubated with UDG and POLβ with 32 P-labeled 34U DNA substrate (1 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing sequencing gel. The non-incised substrate (S), AP site incision product (P1), and gap-filling extension product (P2) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. ( B ) Relative AP site cleavage efficiency. Shown are the averages and standard deviations of 4 independent reactions. ( C ) Relative gap-filling activity. Shown are the average and standard deviation of 4 independent assays.

    Techniques Used: Reconstitution Assay, Purification, Variant Assay, Incubation, Labeling, Sequencing, Activity Assay, Standard Deviation

    3) Product Images from "USER(TM) friendly DNA engineering and cloning method by uracil excision"

    Article Title: USER(TM) friendly DNA engineering and cloning method by uracil excision

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm041

    USER enzyme activity assay. A 34-mer oligonucleotide duplex (10 pmol) containing a single dU paired with a deoxyadenine ( Table 1 ) was incubated with a series of EndoVIII and UDG enzyme mixtures for 15 min at 37°C in a 10 μl of T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 20 μg/ml BSA). The reactions were quenched by the addition of 10 μl of 95% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 10 mM EDTA, pH 11, and the reaction products were analyzedon a 15% TBE-Urea denaturing gel. S, 34-nt oligonucleotide substrate. P1 and P2, 15 nt and 18 nt cleavage products, respectively. Two product bands of differing size result from the fact that the uracil is not in the centre of the substrate. Lane 1—no enzyme added. Lane 2—reaction contains 0.2 units of UDG. Lane 3—reaction contains 256 ng of EndoVIII. Lanes 4–10, reaction contains 0.2 units of UDG and the amount of EndoVIII shown above the respective lanes.
    Figure Legend Snippet: USER enzyme activity assay. A 34-mer oligonucleotide duplex (10 pmol) containing a single dU paired with a deoxyadenine ( Table 1 ) was incubated with a series of EndoVIII and UDG enzyme mixtures for 15 min at 37°C in a 10 μl of T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 20 μg/ml BSA). The reactions were quenched by the addition of 10 μl of 95% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 10 mM EDTA, pH 11, and the reaction products were analyzedon a 15% TBE-Urea denaturing gel. S, 34-nt oligonucleotide substrate. P1 and P2, 15 nt and 18 nt cleavage products, respectively. Two product bands of differing size result from the fact that the uracil is not in the centre of the substrate. Lane 1—no enzyme added. Lane 2—reaction contains 0.2 units of UDG. Lane 3—reaction contains 256 ng of EndoVIII. Lanes 4–10, reaction contains 0.2 units of UDG and the amount of EndoVIII shown above the respective lanes.

    Techniques Used: Enzyme Activity Assay, Incubation

    4) Product Images from "First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G"

    Article Title: First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    Journal: ACS Chemical Biology

    doi: 10.1021/cb200440y

    APOBEC3G inhibitors identified by high throughput screening a) Schematic of the fluorescence-based ssDNA cytosine deamination assay. A3G or A3A deaminates C-to-U, UDG excises the U, NaOH breaks the phosphodiester backbone, and the 5’ fluorophore 6-FAM releases from the 3’ quench TAMRA. The resulting fluorescent read-out directly reports DNA deaminase activity because UDG and NaOH are not rate limiting. b) Representative HTS data. Each X represents a single data point and the red-shaded X’s signify confirmed inhibitors. The first and last two columns of each LOPAC 384 well plate contain non-inhibitory (DMSO) or inhibitory (MN1) controls. c, d, e) Representative A3G inhibitor dose response assays. The indicated concentrations of MN1, MN26, or MN30 were incubated with A3G (green symbols) or A3A (red symbols) in triplicate, and deaminase activity was quantified as above. UDG reactions lacked deaminase protein and used ssDNA substrate with a single uracil. Standard deviations and IC50 values are indicated.
    Figure Legend Snippet: APOBEC3G inhibitors identified by high throughput screening a) Schematic of the fluorescence-based ssDNA cytosine deamination assay. A3G or A3A deaminates C-to-U, UDG excises the U, NaOH breaks the phosphodiester backbone, and the 5’ fluorophore 6-FAM releases from the 3’ quench TAMRA. The resulting fluorescent read-out directly reports DNA deaminase activity because UDG and NaOH are not rate limiting. b) Representative HTS data. Each X represents a single data point and the red-shaded X’s signify confirmed inhibitors. The first and last two columns of each LOPAC 384 well plate contain non-inhibitory (DMSO) or inhibitory (MN1) controls. c, d, e) Representative A3G inhibitor dose response assays. The indicated concentrations of MN1, MN26, or MN30 were incubated with A3G (green symbols) or A3A (red symbols) in triplicate, and deaminase activity was quantified as above. UDG reactions lacked deaminase protein and used ssDNA substrate with a single uracil. Standard deviations and IC50 values are indicated.

    Techniques Used: High Throughput Screening Assay, Fluorescence, Activity Assay, Incubation

    5) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    6) Product Images from "Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification"

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031817

    Selective rolling circle amplification (RCA) flowchart. The heteroduplex, ccc-ds(U)DNA formed in Kunkel mutagenesis (A), is treated with UDG (B) and subsequently amplified with RCA (C) using random hexamers as primers. The resulting DNA concatemer is cut to plasmid-sized units (D) and re-circularized by self-ligation (E) for host transformation.
    Figure Legend Snippet: Selective rolling circle amplification (RCA) flowchart. The heteroduplex, ccc-ds(U)DNA formed in Kunkel mutagenesis (A), is treated with UDG (B) and subsequently amplified with RCA (C) using random hexamers as primers. The resulting DNA concatemer is cut to plasmid-sized units (D) and re-circularized by self-ligation (E) for host transformation.

    Techniques Used: Amplification, Countercurrent Chromatography, Mutagenesis, Plasmid Preparation, Ligation, Transformation Assay

    7) Product Images from "USER(TM) friendly DNA engineering and cloning method by uracil excision"

    Article Title: USER(TM) friendly DNA engineering and cloning method by uracil excision

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm041

    USER enzyme activity assay. A 34-mer oligonucleotide duplex (10 pmol) containing a single dU paired with a deoxyadenine ( Table 1 ) was incubated with a series of EndoVIII and UDG enzyme mixtures for 15 min at 37°C in a 10 μl of T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 20 μg/ml BSA). The reactions were quenched by the addition of 10 μl of 95% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 10 mM EDTA, pH 11, and the reaction products were analyzedon a 15% TBE-Urea denaturing gel. S, 34-nt oligonucleotide substrate. P1 and P2, 15 nt and 18 nt cleavage products, respectively. Two product bands of differing size result from the fact that the uracil is not in the centre of the substrate. Lane 1—no enzyme added. Lane 2—reaction contains 0.2 units of UDG. Lane 3—reaction contains 256 ng of EndoVIII. Lanes 4–10, reaction contains 0.2 units of UDG and the amount of EndoVIII shown above the respective lanes.
    Figure Legend Snippet: USER enzyme activity assay. A 34-mer oligonucleotide duplex (10 pmol) containing a single dU paired with a deoxyadenine ( Table 1 ) was incubated with a series of EndoVIII and UDG enzyme mixtures for 15 min at 37°C in a 10 μl of T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 20 μg/ml BSA). The reactions were quenched by the addition of 10 μl of 95% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 10 mM EDTA, pH 11, and the reaction products were analyzedon a 15% TBE-Urea denaturing gel. S, 34-nt oligonucleotide substrate. P1 and P2, 15 nt and 18 nt cleavage products, respectively. Two product bands of differing size result from the fact that the uracil is not in the centre of the substrate. Lane 1—no enzyme added. Lane 2—reaction contains 0.2 units of UDG. Lane 3—reaction contains 256 ng of EndoVIII. Lanes 4–10, reaction contains 0.2 units of UDG and the amount of EndoVIII shown above the respective lanes.

    Techniques Used: Enzyme Activity Assay, Incubation

    8) Product Images from "Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus"

    Article Title: Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

    Journal: Scientific Reports

    doi: 10.1038/srep27605

    Schematic diagram showing the mechanism of the uracil DNA glycosylase-supplemented real-time reverse-transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) assay. Blue LAMP amplicons represent non-dUTP-incorporated DNA and yellow LAMP amplicons represent dUTP-incorporated DNA. The red enzyme represents Bst 2.0 WarmStart ® DNA polymerase and the blue enzyme represents UDG. UDG-rRT-LAMP eliminates carryover contamination via two steps. ( A ) The first step of the incorporation of dUTP in all LAMP amplicons. ( B ) The second step of UDG-based elimination of carryover contaminants by specifically cutting the LAMP amplicon DNA at the 5′ side of the dUTP-incorporated templates from previous LAMP reactions while having no effect on non-dUTP-incorporated DNA and RNA templates. During the RT-LAMP reaction, the digested contaminants are degraded into small fragments, and UDG is inactivated at approximately 63 °C, ensuring that only the RNA template is amplified.
    Figure Legend Snippet: Schematic diagram showing the mechanism of the uracil DNA glycosylase-supplemented real-time reverse-transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) assay. Blue LAMP amplicons represent non-dUTP-incorporated DNA and yellow LAMP amplicons represent dUTP-incorporated DNA. The red enzyme represents Bst 2.0 WarmStart ® DNA polymerase and the blue enzyme represents UDG. UDG-rRT-LAMP eliminates carryover contamination via two steps. ( A ) The first step of the incorporation of dUTP in all LAMP amplicons. ( B ) The second step of UDG-based elimination of carryover contaminants by specifically cutting the LAMP amplicon DNA at the 5′ side of the dUTP-incorporated templates from previous LAMP reactions while having no effect on non-dUTP-incorporated DNA and RNA templates. During the RT-LAMP reaction, the digested contaminants are degraded into small fragments, and UDG is inactivated at approximately 63 °C, ensuring that only the RNA template is amplified.

    Techniques Used: Amplification, Lamp Assay

    9) Product Images from "Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa"

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.8557

    Sensitivity testing for P. aeruginosa detection in contact lens samples by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.1×10 5 CFU ml −1 to 1.1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.
    Figure Legend Snippet: Sensitivity testing for P. aeruginosa detection in contact lens samples by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.1×10 5 CFU ml −1 to 1.1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Techniques Used: Polymerase Chain Reaction, Serial Dilution, Molecular Weight, Marker, Negative Control, Amplification

    (A) Optimization of hybridization temperatures and durations for the detection of UDG-LAMP products at 57, 62 and 65°C for 5, 10 and 20 min, respectively. Lane G, AuNPs probe only (without salt solution); lane N, negative control (no DNA template). Absorption of ultraviolet-visible spectrophotometer at a wavelength of 525 nm for the detection of UDG-LAMP products using hybridization time at 5, 10 and 20 min for (B) 57°C, (C) 62°C and (D) 65°C. Blue bars indicate positive sample and orange bars indicate negative samples. UDG-LAMP, uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification; AuNPs, gold nanoparticles.
    Figure Legend Snippet: (A) Optimization of hybridization temperatures and durations for the detection of UDG-LAMP products at 57, 62 and 65°C for 5, 10 and 20 min, respectively. Lane G, AuNPs probe only (without salt solution); lane N, negative control (no DNA template). Absorption of ultraviolet-visible spectrophotometer at a wavelength of 525 nm for the detection of UDG-LAMP products using hybridization time at 5, 10 and 20 min for (B) 57°C, (C) 62°C and (D) 65°C. Blue bars indicate positive sample and orange bars indicate negative samples. UDG-LAMP, uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Techniques Used: Hybridization, Negative Control, Spectrophotometry, Amplification

    Sensitivity testing for P. aeruginosa pure culture detection by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.6×10 6 CFU ml −1 to 1.6×10 1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.
    Figure Legend Snippet: Sensitivity testing for P. aeruginosa pure culture detection by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.6×10 6 CFU ml −1 to 1.6×10 1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Techniques Used: Polymerase Chain Reaction, Serial Dilution, Molecular Weight, Marker, Negative Control, Amplification

    Optimization of the MgSO 4 concentration for UDG-LAMP-AuNPs. (A) Various concentrations of MgSO 4 were added with the AuNPs probe to P. aeruginosa UDG-LAMP products and (B) non- P. aeruginosa UDG-LAMP products (negative control). Hybridization was performed at 65°C for 5 min. Lane G, AuNPs probe only (without salt solution); lane 1–6, MgSO 4 concentration at 5, 10, 20, 40, 100 and 200 mM, respectively; lane N, negative control (no DNA template). Absorption spectra of the colloidal AuNP probe in the presence of (C) P. aeruginosa UDG-LAMP products and (D) non- P. aeruginosa UDG-LAMP products under various concentrations of MgSO 4 . (E) Absorption of UV-visible spectrophotometer at the wavelength of 525 nm. Blue bar indicates positive samples and the orange bar indicates negative samples. LAMP, loop-mediated isothermal amplification; UDG-LAMP-AuNPs, uracil-DNA-glycosylase-supplemented LAMP coupled with nanogold probe; AuNPs, gold nanoparticles; PA/ P. aeruginosa, Pseudomonas aeruginosa .
    Figure Legend Snippet: Optimization of the MgSO 4 concentration for UDG-LAMP-AuNPs. (A) Various concentrations of MgSO 4 were added with the AuNPs probe to P. aeruginosa UDG-LAMP products and (B) non- P. aeruginosa UDG-LAMP products (negative control). Hybridization was performed at 65°C for 5 min. Lane G, AuNPs probe only (without salt solution); lane 1–6, MgSO 4 concentration at 5, 10, 20, 40, 100 and 200 mM, respectively; lane N, negative control (no DNA template). Absorption spectra of the colloidal AuNP probe in the presence of (C) P. aeruginosa UDG-LAMP products and (D) non- P. aeruginosa UDG-LAMP products under various concentrations of MgSO 4 . (E) Absorption of UV-visible spectrophotometer at the wavelength of 525 nm. Blue bar indicates positive samples and the orange bar indicates negative samples. LAMP, loop-mediated isothermal amplification; UDG-LAMP-AuNPs, uracil-DNA-glycosylase-supplemented LAMP coupled with nanogold probe; AuNPs, gold nanoparticles; PA/ P. aeruginosa, Pseudomonas aeruginosa .

    Techniques Used: Concentration Assay, Negative Control, Hybridization, Spectrophotometry, Amplification

    10) Product Images from "AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe"

    Article Title: AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr933

    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) Udg activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
    Figure Legend Snippet: Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) Udg activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.

    Techniques Used: Activity Assay, Positive Control, Incubation, Sequencing

    11) Product Images from "Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair"

    Article Title: Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2011.135

    Inhibition of DNA replication induced by pemetrexed. ( a ) Schematic diagram of the CldU, pemetrexed and IdU pulse treatments. ( b ) UDG −/− and ( c ) UDG +/+ cells were fixed at 6 and 24 h after treatments and subjected to fluorescent immunostaining. Replication foci were labeled with CldU (green) and IdU (red). Results are representative of two independent experiments. ( d ) The ratio of IdU to CldU was measured as the ratio of the mean fluorescent density of IdU versus CIdU for each cell ( n =40)
    Figure Legend Snippet: Inhibition of DNA replication induced by pemetrexed. ( a ) Schematic diagram of the CldU, pemetrexed and IdU pulse treatments. ( b ) UDG −/− and ( c ) UDG +/+ cells were fixed at 6 and 24 h after treatments and subjected to fluorescent immunostaining. Replication foci were labeled with CldU (green) and IdU (red). Results are representative of two independent experiments. ( d ) The ratio of IdU to CldU was measured as the ratio of the mean fluorescent density of IdU versus CIdU for each cell ( n =40)

    Techniques Used: Inhibition, Immunostaining, Labeling

    MX-bound AP sites induced by the combination of pemetrexed and MX are lethal DNA lesions. ( a ) The dose-dependent relationship between the levels of AP sites and the concentrations of pemetrexed. The AP sites in DNA were measured using ARP reagent after H460 cells were treated with pemetrexed alone (0–400 nM) or in combination with MX (6 mM) for 24 h. MX-bound AP sites were determined by the differences between AP sites induced by pemetrexed alone and the combination of pemetrexed and MX. ( b ) UDG induction detected in cells treated with the pemetrexed alone and in combination with MX. Cells were collected and UDG protein was measured by western blotting analysis. ( c ) H460 cells grown on the coverslip were treated with pemetrexed alone (100 nM) and in combination with MX (6 mM) for 24 h and subjective to the fluorescent immunostaining. The signal of UDG protein (green) was significantly enhanced and localized in nucleus (blue) of cells treated with the combination of pemetrexed and MX. ( d ) γ H2AX foci formation detected in H460 cells treated with pemetrexed (100 nM) alone and in combination with MX (6 mM) for 24 h; ( e ) Induction of the cleaved PARP and γ H2AX proteins was detected by western blotting in cells with the same treatments
    Figure Legend Snippet: MX-bound AP sites induced by the combination of pemetrexed and MX are lethal DNA lesions. ( a ) The dose-dependent relationship between the levels of AP sites and the concentrations of pemetrexed. The AP sites in DNA were measured using ARP reagent after H460 cells were treated with pemetrexed alone (0–400 nM) or in combination with MX (6 mM) for 24 h. MX-bound AP sites were determined by the differences between AP sites induced by pemetrexed alone and the combination of pemetrexed and MX. ( b ) UDG induction detected in cells treated with the pemetrexed alone and in combination with MX. Cells were collected and UDG protein was measured by western blotting analysis. ( c ) H460 cells grown on the coverslip were treated with pemetrexed alone (100 nM) and in combination with MX (6 mM) for 24 h and subjective to the fluorescent immunostaining. The signal of UDG protein (green) was significantly enhanced and localized in nucleus (blue) of cells treated with the combination of pemetrexed and MX. ( d ) γ H2AX foci formation detected in H460 cells treated with pemetrexed (100 nM) alone and in combination with MX (6 mM) for 24 h; ( e ) Induction of the cleaved PARP and γ H2AX proteins was detected by western blotting in cells with the same treatments

    Techniques Used: Western Blot, Immunostaining

    Cellular response to uracil-DNA induced by pemetrexed. Comparison of cell cycle progression before and after treatment with pemetrexed between ( a ) UDG −/− and ( b ) UDG +/+ cells. Protein alterations in the response to DNA damage, cell cycle progression, and cell death were detected in ( c ) UDG −/− cells and ( d ) UDG +/+ cells treated with pemetrexed. Cells were collected at 6 and 24 h after pemetrexed treatment. Results are representative of two independent experiments
    Figure Legend Snippet: Cellular response to uracil-DNA induced by pemetrexed. Comparison of cell cycle progression before and after treatment with pemetrexed between ( a ) UDG −/− and ( b ) UDG +/+ cells. Protein alterations in the response to DNA damage, cell cycle progression, and cell death were detected in ( c ) UDG −/− cells and ( d ) UDG +/+ cells treated with pemetrexed. Cells were collected at 6 and 24 h after pemetrexed treatment. Results are representative of two independent experiments

    Techniques Used:

    UDG activity determines the levels of uracil and AP sites in DNA. ( a ) UDG activity assay in vitro . Oligonucleotide duplexes containing U:G were incubated with cell extracts (5–10 μ g) from UDG +/+ , DLD1 flag , and UDG −/− cells at 37°C for 1 h. Reaction products were resolved by electrophoresis through denaturing 20% polyacrylamide gels. ( b ) Incorporated uracil detected in UDG +/+ and UDG −/− cells by HPLC/MS/MS analysis. Cells were treated with pemetrexed (10 μ M) for 6, 24, 48, and 72 h. Cells were harvested and 40 μ g of extracted DNA were in vitro reacted with purified UDG (10 U) for 2 h. ( c ) Cells were treated with 5-FU (10 μ M) for 6, 24, 48, and 72 h. Uracil was quantified in the reaction product by LC-MS analysis. ( d ) AP site formed by pemetrexed in UDG +/+ and UDG −/− cells. Cells were treated with pemetrexed (0–400 nM) for 24 h. DNA was extracted and AP sites measured by ARP reagent. ( e ) AP site detected in DNA of UDG −/− cells after reacted with purified UDG in vitro . Cells were treated with pemetrexed (0–100 nM) for 24 h and 40 μ g DNA extracted from cells was in vitro reacted with purified UDG (10 U) for 2 h and AP sites were measured using ARP. Results are representative of three independent experiments
    Figure Legend Snippet: UDG activity determines the levels of uracil and AP sites in DNA. ( a ) UDG activity assay in vitro . Oligonucleotide duplexes containing U:G were incubated with cell extracts (5–10 μ g) from UDG +/+ , DLD1 flag , and UDG −/− cells at 37°C for 1 h. Reaction products were resolved by electrophoresis through denaturing 20% polyacrylamide gels. ( b ) Incorporated uracil detected in UDG +/+ and UDG −/− cells by HPLC/MS/MS analysis. Cells were treated with pemetrexed (10 μ M) for 6, 24, 48, and 72 h. Cells were harvested and 40 μ g of extracted DNA were in vitro reacted with purified UDG (10 U) for 2 h. ( c ) Cells were treated with 5-FU (10 μ M) for 6, 24, 48, and 72 h. Uracil was quantified in the reaction product by LC-MS analysis. ( d ) AP site formed by pemetrexed in UDG +/+ and UDG −/− cells. Cells were treated with pemetrexed (0–400 nM) for 24 h. DNA was extracted and AP sites measured by ARP reagent. ( e ) AP site detected in DNA of UDG −/− cells after reacted with purified UDG in vitro . Cells were treated with pemetrexed (0–100 nM) for 24 h and 40 μ g DNA extracted from cells was in vitro reacted with purified UDG (10 U) for 2 h and AP sites were measured using ARP. Results are representative of three independent experiments

    Techniques Used: Activity Assay, In Vitro, Incubation, Electrophoresis, High Performance Liquid Chromatography, Mass Spectrometry, Purification, Liquid Chromatography

    12) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    Journal: Genes and Environment

    doi: 10.1186/s41021-018-0112-5

    Preparation of DNA template (uracil substrate) with uracil at a defined position. a Upper strand sequence containing uracil base and lower strand substrate containing original C base in pBS2/Uracil are shown diagrammatically. b Experimental procedure for purification of pBS2/uracil using uracil oligo. c Identified DNA substrates (100 ng), pBS2/uracil, were incubated with UDG (0.5 units) or UDG (0.5 units) + APE1 (5 units) at 37 °C for 30 min. Aliquots from the sample were analyzed on 0.8% agarose gel, and the DNA substrates were visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, UDG-treatment; lane 4, UDG + APE1-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows
    Figure Legend Snippet: Preparation of DNA template (uracil substrate) with uracil at a defined position. a Upper strand sequence containing uracil base and lower strand substrate containing original C base in pBS2/Uracil are shown diagrammatically. b Experimental procedure for purification of pBS2/uracil using uracil oligo. c Identified DNA substrates (100 ng), pBS2/uracil, were incubated with UDG (0.5 units) or UDG (0.5 units) + APE1 (5 units) at 37 °C for 30 min. Aliquots from the sample were analyzed on 0.8% agarose gel, and the DNA substrates were visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, UDG-treatment; lane 4, UDG + APE1-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows

    Techniques Used: Sequencing, Purification, Incubation, Agarose Gel Electrophoresis, Staining, Marker, Countercurrent Chromatography

    13) Product Images from "A Fluorescent Probe to Measure DNA Damage and Repair"

    Article Title: A Fluorescent Probe to Measure DNA Damage and Repair

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131330

    Evaluation of base pairing on SSB activity. Time course represents incubation time of U:X DNA before APE addition with UDG and (A) compound 7 or (B) with vehicle control.
    Figure Legend Snippet: Evaluation of base pairing on SSB activity. Time course represents incubation time of U:X DNA before APE addition with UDG and (A) compound 7 or (B) with vehicle control.

    Techniques Used: Activity Assay, Incubation

    Molecular view of normal and interrupted BER. Path A: the short patch BER pathway following UDG removal of uracil in DNA. Path B: the proposed mechanism for interception of the reactive aldehyde present in the AP site with an aminooxy-tagged probe, which blocks further repair by APE and prevents the single strand break (SSB).
    Figure Legend Snippet: Molecular view of normal and interrupted BER. Path A: the short patch BER pathway following UDG removal of uracil in DNA. Path B: the proposed mechanism for interception of the reactive aldehyde present in the AP site with an aminooxy-tagged probe, which blocks further repair by APE and prevents the single strand break (SSB).

    Techniques Used:

    SSB activity assay comparison of ARP, MX, and 7. (A) ARP (1 nmol), MX (1 nmol), and 7 (1 nmol) with UDG (5 units) and U:A DNA (5pmol) as a function of incubation time prior to APE (10 units) addition. (B) ARP (200 nmol), MX (200 nmol), and 7 (2 nmol) with UDG (5 units) and U:A DNA (5 pmol) as a function of incubation time prior to APE (1 unit) addition.
    Figure Legend Snippet: SSB activity assay comparison of ARP, MX, and 7. (A) ARP (1 nmol), MX (1 nmol), and 7 (1 nmol) with UDG (5 units) and U:A DNA (5pmol) as a function of incubation time prior to APE (10 units) addition. (B) ARP (200 nmol), MX (200 nmol), and 7 (2 nmol) with UDG (5 units) and U:A DNA (5 pmol) as a function of incubation time prior to APE (1 unit) addition.

    Techniques Used: Activity Assay, Incubation

    Evaluation of UDG inhibition by 7. Time course represents incubation time of U:A DNA before APE addition with UDG alone, 10 minutes UDG pretreatment then 7 , and UDG and 7 without pretreatment.
    Figure Legend Snippet: Evaluation of UDG inhibition by 7. Time course represents incubation time of U:A DNA before APE addition with UDG alone, 10 minutes UDG pretreatment then 7 , and UDG and 7 without pretreatment.

    Techniques Used: Inhibition, Incubation

    14) Product Images from "First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G"

    Article Title: First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    Journal: ACS Chemical Biology

    doi: 10.1021/cb200440y

    APOBEC3G inhibitors identified by high throughput screening a) Schematic of the fluorescence-based ssDNA cytosine deamination assay. A3G or A3A deaminates C-to-U, UDG excises the U, NaOH breaks the phosphodiester backbone, and the 5’ fluorophore 6-FAM releases from the 3’ quench TAMRA. The resulting fluorescent read-out directly reports DNA deaminase activity because UDG and NaOH are not rate limiting. b) Representative HTS data. Each X represents a single data point and the red-shaded X’s signify confirmed inhibitors. The first and last two columns of each LOPAC 384 well plate contain non-inhibitory (DMSO) or inhibitory (MN1) controls. c, d, e) Representative A3G inhibitor dose response assays. The indicated concentrations of MN1, MN26, or MN30 were incubated with A3G (green symbols) or A3A (red symbols) in triplicate, and deaminase activity was quantified as above. UDG reactions lacked deaminase protein and used ssDNA substrate with a single uracil. Standard deviations and IC50 values are indicated.
    Figure Legend Snippet: APOBEC3G inhibitors identified by high throughput screening a) Schematic of the fluorescence-based ssDNA cytosine deamination assay. A3G or A3A deaminates C-to-U, UDG excises the U, NaOH breaks the phosphodiester backbone, and the 5’ fluorophore 6-FAM releases from the 3’ quench TAMRA. The resulting fluorescent read-out directly reports DNA deaminase activity because UDG and NaOH are not rate limiting. b) Representative HTS data. Each X represents a single data point and the red-shaded X’s signify confirmed inhibitors. The first and last two columns of each LOPAC 384 well plate contain non-inhibitory (DMSO) or inhibitory (MN1) controls. c, d, e) Representative A3G inhibitor dose response assays. The indicated concentrations of MN1, MN26, or MN30 were incubated with A3G (green symbols) or A3A (red symbols) in triplicate, and deaminase activity was quantified as above. UDG reactions lacked deaminase protein and used ssDNA substrate with a single uracil. Standard deviations and IC50 values are indicated.

    Techniques Used: High Throughput Screening Assay, Fluorescence, Activity Assay, Incubation

    15) Product Images from "Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA"

    Article Title: Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024848

    Purification and characterization of proteins used in the study. a. Coomassie-stained 12% SDS-PAGE gel of aliquotes from different steps of A3G purification from HEK293T cells. Mr – molecular weight marker, 1 – clarified lysate, 2 – flow through, 3–8 – different protein-containing fractions, eluted from the resin. b. Deaminase activity of purified A3G, detected in the oligonucleotide assay with uracil-DNA-glycosylase. In this assay, after deaminase converts cytosine to uracil, uracil-DNA-glycosilase removes uracil from the DNA, leading to formation of the AP site, which is further converted into strand break under conditions of high pH and temperature. 1 – UDG alone, 2 - A3G-expressing HEK293T lysate, 3 – clarified lysate, 4 – clarified lysate treated with RNAse, 5 – flow through, 6–11 – different fractions of purified protein. c and d. DNA-binding activity of purified A3G (c) and human RPA (d), detected by electromobility shift assay (EMSA). A3G from fraction 5 (see panel a) was used in this assay. The same oligonucleotide was used for both proteins (c). The band that corresponds to the free oligonucleotide folded to the secondary structure is indicated by the asterisk. Note that in (c) this non-specific band migrates similarly to the fastest A3G-shifted band.
    Figure Legend Snippet: Purification and characterization of proteins used in the study. a. Coomassie-stained 12% SDS-PAGE gel of aliquotes from different steps of A3G purification from HEK293T cells. Mr – molecular weight marker, 1 – clarified lysate, 2 – flow through, 3–8 – different protein-containing fractions, eluted from the resin. b. Deaminase activity of purified A3G, detected in the oligonucleotide assay with uracil-DNA-glycosylase. In this assay, after deaminase converts cytosine to uracil, uracil-DNA-glycosilase removes uracil from the DNA, leading to formation of the AP site, which is further converted into strand break under conditions of high pH and temperature. 1 – UDG alone, 2 - A3G-expressing HEK293T lysate, 3 – clarified lysate, 4 – clarified lysate treated with RNAse, 5 – flow through, 6–11 – different fractions of purified protein. c and d. DNA-binding activity of purified A3G (c) and human RPA (d), detected by electromobility shift assay (EMSA). A3G from fraction 5 (see panel a) was used in this assay. The same oligonucleotide was used for both proteins (c). The band that corresponds to the free oligonucleotide folded to the secondary structure is indicated by the asterisk. Note that in (c) this non-specific band migrates similarly to the fastest A3G-shifted band.

    Techniques Used: Purification, Staining, SDS Page, Molecular Weight, Marker, Flow Cytometry, Activity Assay, Oligonucleotide Assay, Expressing, Binding Assay, Recombinase Polymerase Amplification, Electro Mobility Shift Assay

    16) Product Images from "Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex"

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky620

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.
    Figure Legend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    17) Product Images from "Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining"

    Article Title: Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining

    Journal:

    doi: 10.1016/j.dnarep.2007.07.018

    5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG
    Figure Legend Snippet: 5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG

    Techniques Used: Activity Assay

    Related Articles

    Centrifugation:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: After cell debris was removed by centrifugation (13,000×g , 20 min) at 4°C, supernatant was collected and placed on ice. .. We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Amplification:

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: Primers labeled with Cy5, Cy3, or 6-FAM (6-carboxyfluorescein) were used for PCR. .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. 2.5 U/μL of Circligase II (Biozym 131406) was used and the ligation reaction carried out overnight.

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). .. Completed libraries were quantified with the Agilent Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS Library Quantification kit.

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK). .. Micro Bio-Spin 30 Chromatography Columns were from Bio-Rad.

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
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    Binding Assay:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Oligonucleotide Assay:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. Cleavage reactions were stopped by addition of 20 µl 0.4% fushin in formamide and denaturation at 90°C for 3 min. After quenching on ice, samples were resolved on 17.5% TBE-PAGE urea gels at 200 V and visualised using a Typhoon scanner (GE Healthcare) for fluorescence imaging (Filter: 526 SP (532 nm), Laser: Blue 488 nm).

    Synthesized:

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition
    Article Snippet: Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA). .. Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
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    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Construct:

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Deamination activity was measured using a labeled ssDNA construct 118 nt in length with potential deamination sites 17 nt from the 5′-end and 36 nt from the 3′-end (see Supplementary Fig. for full sequence). .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Real-time Polymerase Chain Reaction:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA.

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
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    Incubation:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: Reactions were stopped and brought to 100 µl total volume in H2 O. .. 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. Cleavage reactions were stopped by addition of 20 µl 0.4% fushin in formamide and denaturation at 90°C for 3 min. After quenching on ice, samples were resolved on 17.5% TBE-PAGE urea gels at 200 V and visualised using a Typhoon scanner (GE Healthcare) for fluorescence imaging (Filter: 526 SP (532 nm), Laser: Blue 488 nm).

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
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    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
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    Article Title: Transient-state kinetics of apurinic/apyrimidinic (AP) endonuclease 1 acting on an authentic AP site and commonly-used substrate analogs: The effect of diverse metal ions and base mismatches
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    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: The labeled DNA was then added at a concentration of 100 nM and incubated for 10 min at 37 °C. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. This treatment fragmented the ssDNA at deamination sites.

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK). .. We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The PCR cycling and melting conditions were as follows; an initial incubation at 95°C for 15 min, followed by 55 cycles of 96°C for 15 s, 70°C for 20 s, 72°C for 30 s; one cycle of 97°C for 1 min and a melt from 70°C to 95°C rising 0.2°C per second. .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The reaction mixture in a final volume of 20 μL was prepared as follows; 1 × PCR buffer, 2.5 mM MgCl2, 400 nM of each primer, 10 ng of FFPE DNA, 200 μM of dNTPs, 5 μM of SYTO9 (Invitrogen), and 0.5 U of HotStarTaq polymerase (Qiagen). .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Activity Assay:

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: Paragraph title: Deamination Assay to Measure Apo3A Specific Activity on ssDNA ... Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Article Title: Specific Inhibition of NEIL-initiated Repair of Oxidized Base Damage in Human Genome by Copper and Iron
    Article Snippet: Paragraph title: Measurement of AP Lyase Activity ... After annealing with the complementary strand, the duplex was treated with 100 units of uracil-DNA glycosylase (New England Biolabs) at 37 °C for 30 min to generate an AP site by excising U.

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Modification:

    Article Title: DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site
    Article Snippet: The integrity of the AP-containing ODN was assessed by PAGE followed by Stains-All staining. .. To confirm the presence of the AP site in the ODN after the treatment with uracil-DNA glycosylase, samples were treated with 10% aqueous piperidine at 95 °C and were completely cleaved at the modified site. .. When needed, the modified strands were 32 P-labeled using [γ-32 P]ATP and phage T4 polynucleotide kinase (SibEnzyme, Novosibirsk, Russia) according to the manufacturer's protocol, purified by 20% denaturing PAGE, and annealed to the complementary strand.

    Hybridization:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    High Performance Liquid Chromatography:

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition
    Article Snippet: Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA). .. Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA).

    Flow Cytometry:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: The lysate-antibody was then incubated with High flow protein-G-Sepharose for 1-2 h at 4°C. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Chromatography:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: .. Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    Ligation:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Buffer Exchange:

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: After incubations for 1, 2, 5, and 10 min, the reactions were quenched by a double extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by a buffer exchange to 10 m m Tris (pH 7.4) using a Micro Bio-spin P-6 column (Bio-Rad). .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Transferring:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: .. Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    Generated:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: We interpret this result as indication that no obvious negative influence on extraction efficiency is caused by using this reduced input bone powder amount. .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. The Klenow Fragment of DNA polymerase I (Thermo Fisher Scientific EP0051) was used for the fill-in reaction [ ].

    Article Title: Specific Inhibition of NEIL-initiated Repair of Oxidized Base Damage in Human Genome by Copper and Iron
    Article Snippet: We generated an AP site-containing duplex oligonucleotide substrate starting with the 51-nt oligonucleotide as described earlier, except for incorporation of U at position 26. .. After annealing with the complementary strand, the duplex was treated with 100 units of uracil-DNA glycosylase (New England Biolabs) at 37 °C for 30 min to generate an AP site by excising U.

    other:

    Article Title: Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage
    Article Snippet: The components are annealed, ligated and the uracil residues converted to abasic sites by uracil-DNA glycosylase (UDG).

    Polymerase Chain Reaction:

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: Primers labeled with Cy5, Cy3, or 6-FAM (6-carboxyfluorescein) were used for PCR. .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). .. Completed libraries were quantified with the Agilent Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS Library Quantification kit.

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK). .. Micro Bio-Spin 30 Chromatography Columns were from Bio-Rad.

    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: Oligonucleotides for PCR primers and substrates containing uracil (U) were purchased from IDT (Coralville, IA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The PCR cycling and melting conditions were as follows; an initial incubation at 95°C for 15 min, followed by 55 cycles of 96°C for 15 s, 70°C for 20 s, 72°C for 30 s; one cycle of 97°C for 1 min and a melt from 70°C to 95°C rising 0.2°C per second. .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix. .. The same PCR conditions were used except an addition of initial incubation at 37°C for 30 min before the activation of HotStarTaq polymerase.

    Sonication:

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: The cell pellet was resuspended in < 5 ml of sonication buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.1 mM DTT), and cells were lysed by sonification. .. We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Recombinant:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: Oligonucleotides were incubated for 30 min at 37°C with 50 ng of recombinant protein. .. 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. All DNAs, with and without gap, were PAGE-purified using a Mini Prep Cell (Bio-Rad, Hercules, CA).

    Article Title: Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene
    Article Snippet: Recombinant human DNA polymerases hPol η (amino acids 1–437), hPol ι (amino acids 1–420), and hPol κ (amino acids 19–526) (active core enzymes) were expressed and purified according to previously published procedures. .. – T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA).

    DNA Extraction:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: DNA extraction was performed according to Dabney et al. [ ] with reduced bone powder input mass, and reduced centrifugation speed of the binding apparatus at approximately 450×g . .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Nucleic Acid Electrophoresis:

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Article Title: Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene
    Article Snippet: Full-length recombinant human polymerase κ (hPol κ ) used for gel electrophoresis experiments was purchased from Enzymax (Lexington, KY). .. – T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA).

    RNA Sequencing Assay:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). .. Completed libraries were quantified with the Agilent Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS Library Quantification kit.

    Fluorescence:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA.

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: After incubations for 1, 2, 5, and 10 min, the reactions were quenched by a double extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by a buffer exchange to 10 m m Tris (pH 7.4) using a Micro Bio-spin P-6 column (Bio-Rad). .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad). .. Products of single deamination at the 5′-side (5′), the 3′-side (3′) target, or double deaminations at both sites (5′ and 3′) were detected as 54, 43, and 32 nt cleaved products, respectively.

    Magnetic Beads:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: Reactions were stopped and brought to 100 µl total volume in H2 O. .. 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. Cleavage reactions were stopped by addition of 20 µl 0.4% fushin in formamide and denaturation at 90°C for 3 min. After quenching on ice, samples were resolved on 17.5% TBE-PAGE urea gels at 200 V and visualised using a Typhoon scanner (GE Healthcare) for fluorescence imaging (Filter: 526 SP (532 nm), Laser: Blue 488 nm).

    Mutagenesis:

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: Paragraph title: Mutation profiling by amplicon sequencing ... For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Isolation:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Labeling:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: PAGE purified deoxyoligonucleotide ( ) was 5′-end 32 P labeled and added into 20 µl of deaminase reaction buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: Primers labeled with Cy5, Cy3, or 6-FAM (6-carboxyfluorescein) were used for PCR. .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ).

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: For reactions at pH 5.1, 5.5, and 6.5, 500 n m labeled ssDNA was incubated with 5 n m Apo3A in the reaction buffer (30 μl) at 37 °C. .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: The labeled DNA was then added at a concentration of 100 nM and incubated for 10 min at 37 °C. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Purification:

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition
    Article Snippet: Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA). .. Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA).

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. The UNG oligonucleotide assay was performed with oligonucleotides 164 (5′-B–ATTATTATTATTAGUTATTTATTTATTTATTTATTTATTT-FITC-3′) and 166 ( 5′-AAATAAATAAATAAATAAATAAATAGCTAATAATAATAAT-3′ ).

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: PAGE purified deoxyoligonucleotide ( ) was 5′-end 32 P labeled and added into 20 µl of deaminase reaction buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. All DNAs, with and without gap, were PAGE-purified using a Mini Prep Cell (Bio-Rad, Hercules, CA).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Article Title: Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene
    Article Snippet: Recombinant human DNA polymerases hPol η (amino acids 1–437), hPol ι (amino acids 1–420), and hPol κ (amino acids 19–526) (active core enzymes) were expressed and purified according to previously published procedures. .. – T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA).

    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Sequencing:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: We interpret this result as indication that no obvious negative influence on extraction efficiency is caused by using this reduced input bone powder amount. .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. The Klenow Fragment of DNA polymerase I (Thermo Fisher Scientific EP0051) was used for the fill-in reaction [ ].

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Deamination activity was measured using a labeled ssDNA construct 118 nt in length with potential deamination sites 17 nt from the 5′-end and 36 nt from the 3′-end (see Supplementary Fig. for full sequence). .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: Paragraph title: Mutation profiling by amplicon sequencing ... For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: PAGE purified deoxyoligonucleotide ( ) was 5′-end 32 P labeled and added into 20 µl of deaminase reaction buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: After incubations for 1, 2, 5, and 10 min, the reactions were quenched by a double extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by a buffer exchange to 10 m m Tris (pH 7.4) using a Micro Bio-spin P-6 column (Bio-Rad). .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad). .. Products of single deamination at the 5′-side (5′), the 3′-side (3′) target, or double deaminations at both sites (5′ and 3′) were detected as 54, 43, and 32 nt cleaved products, respectively.

    IA:

    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: Oligonucleotides for PCR primers and substrates containing uracil (U) were purchased from IDT (Coralville, IA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Mouse Assay:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    HRM Assay:

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix. .. A PIK3CA E549D mutation detected at a low frequency (4%) in the Core 2 of Tumor 2 by AmpliSeq was analysed by limited copy number (LCN)-HRM [ ].

    Plasmid Preparation:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C. .. For in vitro synthesis of A3A and mutants we employed the TNT Coupled Wheat Germ Extract System (Promega) using T7 polymerase.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: DNA for nucleosome preparations was PCR amplified from a plasmid containing the Widom's 601 DNA ( ). .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ).

    Software:

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl.

    SYBR Green Assay:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA. .. Seal the plate with a plastic seal (Bio-Rad catalog# MSB1001), and use the following PCR protocol: 37°C 10 min 95°C 10 min 95°C 30 s 60°C 30 s 74°C 10 s Read plate.

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. A quantitative PCR (qPCR) experiment was carried out using 0.2% of the unamplified library to estimate relative library complexities (Additional file : Table S1), and to determine the optimal number of cycles for subsequent indexing PCR, representing the inflection point of the respective library amplification curves, corrected for reaction volume and template amount. qPCR was performed on a PikoReal 96 Real-Time PCR machine (Thermo Fisher Scientific TCR0096) with 3 replicates for each library, involving an initial 10 min denaturation at 95 °C, followed by 40 cycles of: 15 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C.

    Selection:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    In Vitro:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: Paragraph title: In vitro Deaminase Assays ... The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: Paragraph title: In vitro base-excision repair (BER) assay ... We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Irradiation:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA. .. Seal the plate with a plastic seal (Bio-Rad catalog# MSB1001), and use the following PCR protocol: 37°C 10 min 95°C 10 min 95°C 30 s 60°C 30 s 74°C 10 s Read plate.

    Produced:

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Concentration Assay:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Store your dilutions at 4°C until validated as freeze–thaw cycles alter the effective DNA concentration. .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA.

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: For reactions at pH 7.4 and 8.0, a higher concentration (50 n m ) of Apo3A was used. .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Article Title: Transient-state kinetics of apurinic/apyrimidinic (AP) endonuclease 1 acting on an authentic AP site and commonly-used substrate analogs: The effect of diverse metal ions and base mismatches
    Article Snippet: Assembly of the 30-mer duplex substrate was achieved by annealing 30 pmol (for transient-state experiments) or 300 pmol (for steady-state experiments) of the 5′-radiolabeled lesion-containing strand in the presence of a 1.5-fold excess of the desired complement [either well matched (WM) or containing a mismatch (MM1, MM2, MM3, or MM4)] in 300 μL of 50 mM HEPES-KOH, 100 mM KCl, pH 7.5. .. To generate a duplex containing the reduced AP site, the uracil-containing duplex was incubated with 1.5 units of uracil DNA glycosylase (UDG; New England Biolabs) and freshly prepared NaBH4 (final concentration of NaBH4 was 0.1 M) overnight at 37 °C. .. The DNA was desalted using a 0.5 mL, 3,000 MW Amicon centrifugal filter.

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: The labeled DNA was then added at a concentration of 100 nM and incubated for 10 min at 37 °C. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Lysis:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: The resin was washed three times with lysis buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Variant Assay:

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The HRM primer sequences for FGFR3 exon 6 variant analysis were F 5′-CAGTGGCGGTGGTGGTGAGG-3′ and R 5′-ACCTTGCAGTGGAACTCCACGTC-3′. .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Gas Chromatography-Mass Spectrometry:

    Article Title: Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine, and DNA uracil concentrations
    Article Snippet: Specifically, persons taking certain antipurines (azathioprine), chemotherapy drugs (methotrexate), or antibiotics (sulfamethoxazole and trimethoprim) were excluded. .. DNA was extracted from 1 mL whole frozen blood by using standard phenol:chloroform extraction after proteinase K and RNase treatment; 5.0–10 μ g DNA was used to determine the uracil content after uracil DNA glycosylase (New England Biolabs, Ipswich, MA) treatment according to the gas chromatography–mass spectrometry method of Blount and Ames ( ) with modifications ( ). .. Sufficient DNA for uracil analysis was recovered from 255 individuals.

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    New England Biolabs udg
    Sensitivity testing for P. aeruginosa detection in contact lens samples by (A) <t>UDG-LAMP,</t> (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.1×10 5 CFU ml −1 to 1.1 CFU ml −1 . Lane M, molecular weight <t>DNA</t> marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.
    Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs e coli udg
    <t>HHV-1,</t> human and E. coli <t>UDG</t> are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.
    E Coli Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs hhv 1 udg
    <t>HHV-1,</t> human and E. coli <t>UDG</t> are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.
    Hhv 1 Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs udg assay buffer
    <t>HHV-1,</t> human and E. coli <t>UDG</t> are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.
    Udg Assay Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sensitivity testing for P. aeruginosa detection in contact lens samples by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.1×10 5 CFU ml −1 to 1.1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Journal: Molecular Medicine Reports

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    doi: 10.3892/mmr.2018.8557

    Figure Lengend Snippet: Sensitivity testing for P. aeruginosa detection in contact lens samples by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.1×10 5 CFU ml −1 to 1.1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Article Snippet: The UDG-LAMP assay was carried out in a total of 25 µl reaction mixture consisted of 40 pmol each of the inner primers (FIP and BIP), 5 pmol each of the outer primers (F3 and B3), 1.4 mM each of dATP, dGTP, dCTP and dUTP: dTTP (different ratios at 100% dUTP; 80% dUTP + 20% dTTP; 60% dUTP + 40% dTTP; 40% dUTP + 60% dTTP; 20% dUTP + 80% dTTP; and 100% dTTP) (New England Biolabs, Inc., Ipswich, MA, USA), 5.0 mM of MgSO4 , 0.8 M Betaine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 8 U of Bst 2.0 DNA polymerase (New England Biolabs), 5 U of UDG (New England Biolabs), 1X of supplied buffer and DNA template.

    Techniques: Polymerase Chain Reaction, Serial Dilution, Molecular Weight, Marker, Negative Control, Amplification

    (A) Optimization of hybridization temperatures and durations for the detection of UDG-LAMP products at 57, 62 and 65°C for 5, 10 and 20 min, respectively. Lane G, AuNPs probe only (without salt solution); lane N, negative control (no DNA template). Absorption of ultraviolet-visible spectrophotometer at a wavelength of 525 nm for the detection of UDG-LAMP products using hybridization time at 5, 10 and 20 min for (B) 57°C, (C) 62°C and (D) 65°C. Blue bars indicate positive sample and orange bars indicate negative samples. UDG-LAMP, uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Journal: Molecular Medicine Reports

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    doi: 10.3892/mmr.2018.8557

    Figure Lengend Snippet: (A) Optimization of hybridization temperatures and durations for the detection of UDG-LAMP products at 57, 62 and 65°C for 5, 10 and 20 min, respectively. Lane G, AuNPs probe only (without salt solution); lane N, negative control (no DNA template). Absorption of ultraviolet-visible spectrophotometer at a wavelength of 525 nm for the detection of UDG-LAMP products using hybridization time at 5, 10 and 20 min for (B) 57°C, (C) 62°C and (D) 65°C. Blue bars indicate positive sample and orange bars indicate negative samples. UDG-LAMP, uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Article Snippet: The UDG-LAMP assay was carried out in a total of 25 µl reaction mixture consisted of 40 pmol each of the inner primers (FIP and BIP), 5 pmol each of the outer primers (F3 and B3), 1.4 mM each of dATP, dGTP, dCTP and dUTP: dTTP (different ratios at 100% dUTP; 80% dUTP + 20% dTTP; 60% dUTP + 40% dTTP; 40% dUTP + 60% dTTP; 20% dUTP + 80% dTTP; and 100% dTTP) (New England Biolabs, Inc., Ipswich, MA, USA), 5.0 mM of MgSO4 , 0.8 M Betaine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 8 U of Bst 2.0 DNA polymerase (New England Biolabs), 5 U of UDG (New England Biolabs), 1X of supplied buffer and DNA template.

    Techniques: Hybridization, Negative Control, Spectrophotometry, Amplification

    Sensitivity testing for P. aeruginosa pure culture detection by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.6×10 6 CFU ml −1 to 1.6×10 1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Journal: Molecular Medicine Reports

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    doi: 10.3892/mmr.2018.8557

    Figure Lengend Snippet: Sensitivity testing for P. aeruginosa pure culture detection by (A) UDG-LAMP, (B) polymerase chain reaction and (C) UDG-LAMP-AuNP. Lanes 1–6 represent the 10-fold serial dilution of P. aeruginosa from 1.6×10 6 CFU ml −1 to 1.6×10 1 CFU ml −1 . Lane M, molecular weight DNA marker; lane N, negative control; lane G, gold nanoparticle probe only; P. aeruginosa, Pseudomonas aeruginosa ; CFU, colony-forming units; UDG, uracil-DNA-glycosylase; LAMP, loop-mediated isothermal amplification; AuNPs, gold nanoparticles.

    Article Snippet: The UDG-LAMP assay was carried out in a total of 25 µl reaction mixture consisted of 40 pmol each of the inner primers (FIP and BIP), 5 pmol each of the outer primers (F3 and B3), 1.4 mM each of dATP, dGTP, dCTP and dUTP: dTTP (different ratios at 100% dUTP; 80% dUTP + 20% dTTP; 60% dUTP + 40% dTTP; 40% dUTP + 60% dTTP; 20% dUTP + 80% dTTP; and 100% dTTP) (New England Biolabs, Inc., Ipswich, MA, USA), 5.0 mM of MgSO4 , 0.8 M Betaine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 8 U of Bst 2.0 DNA polymerase (New England Biolabs), 5 U of UDG (New England Biolabs), 1X of supplied buffer and DNA template.

    Techniques: Polymerase Chain Reaction, Serial Dilution, Molecular Weight, Marker, Negative Control, Amplification

    Optimization of the MgSO 4 concentration for UDG-LAMP-AuNPs. (A) Various concentrations of MgSO 4 were added with the AuNPs probe to P. aeruginosa UDG-LAMP products and (B) non- P. aeruginosa UDG-LAMP products (negative control). Hybridization was performed at 65°C for 5 min. Lane G, AuNPs probe only (without salt solution); lane 1–6, MgSO 4 concentration at 5, 10, 20, 40, 100 and 200 mM, respectively; lane N, negative control (no DNA template). Absorption spectra of the colloidal AuNP probe in the presence of (C) P. aeruginosa UDG-LAMP products and (D) non- P. aeruginosa UDG-LAMP products under various concentrations of MgSO 4 . (E) Absorption of UV-visible spectrophotometer at the wavelength of 525 nm. Blue bar indicates positive samples and the orange bar indicates negative samples. LAMP, loop-mediated isothermal amplification; UDG-LAMP-AuNPs, uracil-DNA-glycosylase-supplemented LAMP coupled with nanogold probe; AuNPs, gold nanoparticles; PA/ P. aeruginosa, Pseudomonas aeruginosa .

    Journal: Molecular Medicine Reports

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    doi: 10.3892/mmr.2018.8557

    Figure Lengend Snippet: Optimization of the MgSO 4 concentration for UDG-LAMP-AuNPs. (A) Various concentrations of MgSO 4 were added with the AuNPs probe to P. aeruginosa UDG-LAMP products and (B) non- P. aeruginosa UDG-LAMP products (negative control). Hybridization was performed at 65°C for 5 min. Lane G, AuNPs probe only (without salt solution); lane 1–6, MgSO 4 concentration at 5, 10, 20, 40, 100 and 200 mM, respectively; lane N, negative control (no DNA template). Absorption spectra of the colloidal AuNP probe in the presence of (C) P. aeruginosa UDG-LAMP products and (D) non- P. aeruginosa UDG-LAMP products under various concentrations of MgSO 4 . (E) Absorption of UV-visible spectrophotometer at the wavelength of 525 nm. Blue bar indicates positive samples and the orange bar indicates negative samples. LAMP, loop-mediated isothermal amplification; UDG-LAMP-AuNPs, uracil-DNA-glycosylase-supplemented LAMP coupled with nanogold probe; AuNPs, gold nanoparticles; PA/ P. aeruginosa, Pseudomonas aeruginosa .

    Article Snippet: The UDG-LAMP assay was carried out in a total of 25 µl reaction mixture consisted of 40 pmol each of the inner primers (FIP and BIP), 5 pmol each of the outer primers (F3 and B3), 1.4 mM each of dATP, dGTP, dCTP and dUTP: dTTP (different ratios at 100% dUTP; 80% dUTP + 20% dTTP; 60% dUTP + 40% dTTP; 40% dUTP + 60% dTTP; 20% dUTP + 80% dTTP; and 100% dTTP) (New England Biolabs, Inc., Ipswich, MA, USA), 5.0 mM of MgSO4 , 0.8 M Betaine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 8 U of Bst 2.0 DNA polymerase (New England Biolabs), 5 U of UDG (New England Biolabs), 1X of supplied buffer and DNA template.

    Techniques: Concentration Assay, Negative Control, Hybridization, Spectrophotometry, Amplification

    HHV-1, human and E. coli UDG are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: HHV-1, human and E. coli UDG are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.

    Article Snippet: UDG assays were performed with HHV-1 UDG and quality controlled against a commercially obtained E. coli UDG (NEB).

    Techniques: Sequencing, Inhibition

    Comparison of UDG-DNA backbone phosphate interactions versus UDG with the inhibitory helix of p56. The UDG carbon atoms are shown in blue, whereas DNA and inhibitor carbon atoms are in yellow. Numbering of UDG residues in left hand side panels (A,C,E,G) refer to human UDG, whereas numbering in right hand side panels (B,D,F,H) refer to HHV-1 UDG; these are equivalent views of the two enzymes. ( A/B ) Area 1 base upstream of the catalysed uracil base compared with the equivalent region with the p56 inhibitor bound. ( C/D ) Region containing the phosphate of the catalysed uracil compared with that with p56 bound. ( E/F ) The phosphate 1 base downstream of the catalysed uracil compared with that with p56 bound. ( G/H ) The final phosphate with significant interactions and the equivalent region with p56 bound.

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: Comparison of UDG-DNA backbone phosphate interactions versus UDG with the inhibitory helix of p56. The UDG carbon atoms are shown in blue, whereas DNA and inhibitor carbon atoms are in yellow. Numbering of UDG residues in left hand side panels (A,C,E,G) refer to human UDG, whereas numbering in right hand side panels (B,D,F,H) refer to HHV-1 UDG; these are equivalent views of the two enzymes. ( A/B ) Area 1 base upstream of the catalysed uracil base compared with the equivalent region with the p56 inhibitor bound. ( C/D ) Region containing the phosphate of the catalysed uracil compared with that with p56 bound. ( E/F ) The phosphate 1 base downstream of the catalysed uracil compared with that with p56 bound. ( G/H ) The final phosphate with significant interactions and the equivalent region with p56 bound.

    Article Snippet: UDG assays were performed with HHV-1 UDG and quality controlled against a commercially obtained E. coli UDG (NEB).

    Techniques:

    Comparative orientations of UDG bound to p56, DNA, and ugi, with detailed views of the UDG-binding pocket containing both substrate and inhibitor elements on a UDG surface. UDG residues are shown with blue carbon atoms as is the surface, whereas substrate and inhibitor residues are shown with yellow carbon atoms. The second dimer of p56 is shown in green. ( A ) The p56 inhibitory helix bound to the HHV-1 UDG. Only the interacting side chains of the inhibitory helix and Glu26 of p56 are shown alongside UDG interacting residues. ( B ) Backbone of the uracil-containing strand of DNA bound to human UDG (PDB: 1SSP). ( C ) Residues of the ugi inhibitory strand bound to human UDG (PDB: 1UGH).

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: Comparative orientations of UDG bound to p56, DNA, and ugi, with detailed views of the UDG-binding pocket containing both substrate and inhibitor elements on a UDG surface. UDG residues are shown with blue carbon atoms as is the surface, whereas substrate and inhibitor residues are shown with yellow carbon atoms. The second dimer of p56 is shown in green. ( A ) The p56 inhibitory helix bound to the HHV-1 UDG. Only the interacting side chains of the inhibitory helix and Glu26 of p56 are shown alongside UDG interacting residues. ( B ) Backbone of the uracil-containing strand of DNA bound to human UDG (PDB: 1SSP). ( C ) Residues of the ugi inhibitory strand bound to human UDG (PDB: 1UGH).

    Article Snippet: UDG assays were performed with HHV-1 UDG and quality controlled against a commercially obtained E. coli UDG (NEB).

    Techniques: Binding Assay

    HHV-1 UDG (molecule A, in main text; shown here in light blue) in complex with PZA p56 [PDB accession 4L5N]. The subunits of p56 are coloured to support the descriptions in the text: molecule C (shown here in yellow and brown) makes the most extensive interactions via its docked helix (the brown segment), which includes forming two sides of a hydrophobic pit that traps UDG leucine 214 (see main text for details), whereas molecule D, as well as forming the remaining two sides of the hydrophobic pit, makes just one other notable interaction with UDG (see main text).

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: HHV-1 UDG (molecule A, in main text; shown here in light blue) in complex with PZA p56 [PDB accession 4L5N]. The subunits of p56 are coloured to support the descriptions in the text: molecule C (shown here in yellow and brown) makes the most extensive interactions via its docked helix (the brown segment), which includes forming two sides of a hydrophobic pit that traps UDG leucine 214 (see main text for details), whereas molecule D, as well as forming the remaining two sides of the hydrophobic pit, makes just one other notable interaction with UDG (see main text).

    Article Snippet: UDG assays were performed with HHV-1 UDG and quality controlled against a commercially obtained E. coli UDG (NEB).

    Techniques:

    HHV-1, human and E. coli UDG are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: HHV-1, human and E. coli UDG are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques: Sequencing, Inhibition

    Comparison of UDG-DNA backbone phosphate interactions versus UDG with the inhibitory helix of p56. The UDG carbon atoms are shown in blue, whereas DNA and inhibitor carbon atoms are in yellow. Numbering of UDG residues in left hand side panels (A,C,E,G) refer to human UDG, whereas numbering in right hand side panels (B,D,F,H) refer to HHV-1 UDG; these are equivalent views of the two enzymes. ( A/B ) Area 1 base upstream of the catalysed uracil base compared with the equivalent region with the p56 inhibitor bound. ( C/D ) Region containing the phosphate of the catalysed uracil compared with that with p56 bound. ( E/F ) The phosphate 1 base downstream of the catalysed uracil compared with that with p56 bound. ( G/H ) The final phosphate with significant interactions and the equivalent region with p56 bound.

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: Comparison of UDG-DNA backbone phosphate interactions versus UDG with the inhibitory helix of p56. The UDG carbon atoms are shown in blue, whereas DNA and inhibitor carbon atoms are in yellow. Numbering of UDG residues in left hand side panels (A,C,E,G) refer to human UDG, whereas numbering in right hand side panels (B,D,F,H) refer to HHV-1 UDG; these are equivalent views of the two enzymes. ( A/B ) Area 1 base upstream of the catalysed uracil base compared with the equivalent region with the p56 inhibitor bound. ( C/D ) Region containing the phosphate of the catalysed uracil compared with that with p56 bound. ( E/F ) The phosphate 1 base downstream of the catalysed uracil compared with that with p56 bound. ( G/H ) The final phosphate with significant interactions and the equivalent region with p56 bound.

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques:

    Comparative orientations of UDG bound to p56, DNA, and ugi, with detailed views of the UDG-binding pocket containing both substrate and inhibitor elements on a UDG surface. UDG residues are shown with blue carbon atoms as is the surface, whereas substrate and inhibitor residues are shown with yellow carbon atoms. The second dimer of p56 is shown in green. ( A ) The p56 inhibitory helix bound to the HHV-1 UDG. Only the interacting side chains of the inhibitory helix and Glu26 of p56 are shown alongside UDG interacting residues. ( B ) Backbone of the uracil-containing strand of DNA bound to human UDG (PDB: 1SSP). ( C ) Residues of the ugi inhibitory strand bound to human UDG (PDB: 1UGH).

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: Comparative orientations of UDG bound to p56, DNA, and ugi, with detailed views of the UDG-binding pocket containing both substrate and inhibitor elements on a UDG surface. UDG residues are shown with blue carbon atoms as is the surface, whereas substrate and inhibitor residues are shown with yellow carbon atoms. The second dimer of p56 is shown in green. ( A ) The p56 inhibitory helix bound to the HHV-1 UDG. Only the interacting side chains of the inhibitory helix and Glu26 of p56 are shown alongside UDG interacting residues. ( B ) Backbone of the uracil-containing strand of DNA bound to human UDG (PDB: 1SSP). ( C ) Residues of the ugi inhibitory strand bound to human UDG (PDB: 1UGH).

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques: Binding Assay

    HHV-1 UDG (molecule A, in main text; shown here in light blue) in complex with PZA p56 [PDB accession 4L5N]. The subunits of p56 are coloured to support the descriptions in the text: molecule C (shown here in yellow and brown) makes the most extensive interactions via its docked helix (the brown segment), which includes forming two sides of a hydrophobic pit that traps UDG leucine 214 (see main text for details), whereas molecule D, as well as forming the remaining two sides of the hydrophobic pit, makes just one other notable interaction with UDG (see main text).

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: HHV-1 UDG (molecule A, in main text; shown here in light blue) in complex with PZA p56 [PDB accession 4L5N]. The subunits of p56 are coloured to support the descriptions in the text: molecule C (shown here in yellow and brown) makes the most extensive interactions via its docked helix (the brown segment), which includes forming two sides of a hydrophobic pit that traps UDG leucine 214 (see main text for details), whereas molecule D, as well as forming the remaining two sides of the hydrophobic pit, makes just one other notable interaction with UDG (see main text).

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques:

    HHV-1, human and E. coli UDG are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: HHV-1, human and E. coli UDG are among the most completely characterized UDGs, including structural data of their association with the PBS1 encoded stoichiometric inhibitor, ugi. Bacillus subtilis UDG is the natural target of the inhibitors ugi and p56. Sequence identity between these UDGs is highlighted in cyan. Positions marked with a gold triangle show important points of contact between HHV-1 UDG and p56 observed in this study, which are also seen in contacts between distorted B-DNA with human UDG-DNA in structure 1SSP ( Figure 3 , and Supplementary Table S1 ). MUG ( E. coli ), TDG (human) and SMUG ( Xenopus ) are other enzymes from the wider UDG superfamily, but important residues in ugi and p56 inhibition are not conserved, these are indicated by aligning only these residues; those residues that are conserved between these enzymes and family 1 UDGs shown, are indicated by a circle above. The orange highlighted region is the UDG minor groove intercalation loop apical hydrophobic residue (phenylalanine in B. subtilis , leucine in other UDGs shown) sequestered by ugi and p56. The presence of an arginine at this position in SMUG and TDG suggests lack of susceptibility to ugi or p56 (see main text). Residue substitutions across the much more highly conserved family 1 UDGs can affect attributes such as turnover and dissociation kinetics, and therefore might also modulate interactions with inhibitor proteins.

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques: Sequencing, Inhibition

    Comparison of UDG-DNA backbone phosphate interactions versus UDG with the inhibitory helix of p56. The UDG carbon atoms are shown in blue, whereas DNA and inhibitor carbon atoms are in yellow. Numbering of UDG residues in left hand side panels (A,C,E,G) refer to human UDG, whereas numbering in right hand side panels (B,D,F,H) refer to HHV-1 UDG; these are equivalent views of the two enzymes. ( A/B ) Area 1 base upstream of the catalysed uracil base compared with the equivalent region with the p56 inhibitor bound. ( C/D ) Region containing the phosphate of the catalysed uracil compared with that with p56 bound. ( E/F ) The phosphate 1 base downstream of the catalysed uracil compared with that with p56 bound. ( G/H ) The final phosphate with significant interactions and the equivalent region with p56 bound.

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: Comparison of UDG-DNA backbone phosphate interactions versus UDG with the inhibitory helix of p56. The UDG carbon atoms are shown in blue, whereas DNA and inhibitor carbon atoms are in yellow. Numbering of UDG residues in left hand side panels (A,C,E,G) refer to human UDG, whereas numbering in right hand side panels (B,D,F,H) refer to HHV-1 UDG; these are equivalent views of the two enzymes. ( A/B ) Area 1 base upstream of the catalysed uracil base compared with the equivalent region with the p56 inhibitor bound. ( C/D ) Region containing the phosphate of the catalysed uracil compared with that with p56 bound. ( E/F ) The phosphate 1 base downstream of the catalysed uracil compared with that with p56 bound. ( G/H ) The final phosphate with significant interactions and the equivalent region with p56 bound.

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques:

    Comparative orientations of UDG bound to p56, DNA, and ugi, with detailed views of the UDG-binding pocket containing both substrate and inhibitor elements on a UDG surface. UDG residues are shown with blue carbon atoms as is the surface, whereas substrate and inhibitor residues are shown with yellow carbon atoms. The second dimer of p56 is shown in green. ( A ) The p56 inhibitory helix bound to the HHV-1 UDG. Only the interacting side chains of the inhibitory helix and Glu26 of p56 are shown alongside UDG interacting residues. ( B ) Backbone of the uracil-containing strand of DNA bound to human UDG (PDB: 1SSP). ( C ) Residues of the ugi inhibitory strand bound to human UDG (PDB: 1UGH).

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: Comparative orientations of UDG bound to p56, DNA, and ugi, with detailed views of the UDG-binding pocket containing both substrate and inhibitor elements on a UDG surface. UDG residues are shown with blue carbon atoms as is the surface, whereas substrate and inhibitor residues are shown with yellow carbon atoms. The second dimer of p56 is shown in green. ( A ) The p56 inhibitory helix bound to the HHV-1 UDG. Only the interacting side chains of the inhibitory helix and Glu26 of p56 are shown alongside UDG interacting residues. ( B ) Backbone of the uracil-containing strand of DNA bound to human UDG (PDB: 1SSP). ( C ) Residues of the ugi inhibitory strand bound to human UDG (PDB: 1UGH).

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques: Binding Assay

    HHV-1 UDG (molecule A, in main text; shown here in light blue) in complex with PZA p56 [PDB accession 4L5N]. The subunits of p56 are coloured to support the descriptions in the text: molecule C (shown here in yellow and brown) makes the most extensive interactions via its docked helix (the brown segment), which includes forming two sides of a hydrophobic pit that traps UDG leucine 214 (see main text for details), whereas molecule D, as well as forming the remaining two sides of the hydrophobic pit, makes just one other notable interaction with UDG (see main text).

    Journal: Nucleic Acids Research

    Article Title: Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

    doi: 10.1093/nar/gkt633

    Figure Lengend Snippet: HHV-1 UDG (molecule A, in main text; shown here in light blue) in complex with PZA p56 [PDB accession 4L5N]. The subunits of p56 are coloured to support the descriptions in the text: molecule C (shown here in yellow and brown) makes the most extensive interactions via its docked helix (the brown segment), which includes forming two sides of a hydrophobic pit that traps UDG leucine 214 (see main text for details), whereas molecule D, as well as forming the remaining two sides of the hydrophobic pit, makes just one other notable interaction with UDG (see main text).

    Article Snippet: Reactions were performed at 37°C for 30 min in total volumes of 24 µl [1× NEB buffer #3 for HHV-1 UDG, 1× NEB buffer #1 for HsSMUG, 1× UDG assay buffer (NEB) for E. coli UDG] incorporating 5 units of Endonuclease IV (Nfo) (NEB), which was also the case for control DNA samples.

    Techniques: