udg  (New England Biolabs)


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    Name:
    Uracil DNA Glycosylase UDG
    Description:
    Uracil DNA Glycosylase UDG 5 000 units
    Catalog Number:
    m0280l
    Price:
    301
    Size:
    5 000 units
    Category:
    DNA Glycosylases
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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 45 article reviews
    Price from $9.99 to $1999.99
    udg - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Acetylation of the histone H3 tail domain regulates base excision repair on higher-order chromatin structures"

    Article Title: Acetylation of the histone H3 tail domain regulates base excision repair on higher-order chromatin structures

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52340-0

    Acetylation of histone H3 differentially regulates UDG/APE1-mediated removal of dU49 on compact chromatin. The indicated substrates (10 nM; Fig. 1 ) were incubated with UDG and APE1 (1 nM each) in the presence of 2.0 mM Mg 2+ for 60 minutes. Error bars represent standard deviation from at least three independent experiments (*P
    Figure Legend Snippet: Acetylation of histone H3 differentially regulates UDG/APE1-mediated removal of dU49 on compact chromatin. The indicated substrates (10 nM; Fig. 1 ) were incubated with UDG and APE1 (1 nM each) in the presence of 2.0 mM Mg 2+ for 60 minutes. Error bars represent standard deviation from at least three independent experiments (*P

    Techniques Used: Incubation, Standard Deviation

    BER of damaged DNA within various chromatin environments. The indicated substrates (10 nM; Fig. 1 ) were incubated with UDG and APE1 (1 nM each) in the presence of either 0.2 mM Mg 2+ ( a,b ) or 2.0 mM Mg 2+ ( c,d ). Error bars represent standard deviation from at least three independent experiments. See Table 1 for relevant statistics.
    Figure Legend Snippet: BER of damaged DNA within various chromatin environments. The indicated substrates (10 nM; Fig. 1 ) were incubated with UDG and APE1 (1 nM each) in the presence of either 0.2 mM Mg 2+ ( a,b ) or 2.0 mM Mg 2+ ( c,d ). Error bars represent standard deviation from at least three independent experiments. See Table 1 for relevant statistics.

    Techniques Used: Incubation, Standard Deviation

    2) 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

    3) Product Images from "Isolation and analysis of rereplicated DNA by Rerep-Seq"

    Article Title: Isolation and analysis of rereplicated DNA by Rerep-Seq

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa197

    Fundamentals of Rerep-Seq. ( A ) Schematic of selective fragmentation of rereplicated DNA. Replicating DNA incorporates BrdU into newly synthesized strand, rereplicating DNA acquires BrdU in both strands. Rerep-Seq digest: UVA exposure photolyzes incorporated BrdU to produce uracil, followed by nucleotide excision by UDG, to produce an abasic site and generation of single strand breaks through digestion with APE1. Normally replicated DNA generates single stranded breaks and staggered double stranded breaks in rereplicated DNA. ( B and C ) Selective fragmentation of yeast (B) and human (C) genomic DNA requires BrdU, UVA and UDG/APE-1 digestion. Genomic DNA from cells labeled with BrdU for zero or two cell cycles (−,+ BrdU) to mimic double labeled rereplicated DNA was treated with the indicated steps of the Rerep-Seq digest procedure. ( D and E ) Optimization of fragment size for yeast and human DNA. Genomic DNA exposed to varying doses of UVA followed by treatment with UGD/APE1, and DNA exposed to UVA followed by varying UDG and APE1 digestion time. ( F and G ) Selective fragmentation and enrichment of double BrdU labeled yeast DNA. ( H and I ) Selective fragmentation and enrichment of double BrdU labeled human DNA. Rerep-digest on genomic DNA treated with BrdU for 1, 2 or 3 cell cycles. DNA fragments were gel extracted and equal volumes were analyzed by qPCR with primers within ARS307 (yeast) and ACTb gene (human). Data represent the average of three biological replicates ( n = 3), error bars represent the SEM.
    Figure Legend Snippet: Fundamentals of Rerep-Seq. ( A ) Schematic of selective fragmentation of rereplicated DNA. Replicating DNA incorporates BrdU into newly synthesized strand, rereplicating DNA acquires BrdU in both strands. Rerep-Seq digest: UVA exposure photolyzes incorporated BrdU to produce uracil, followed by nucleotide excision by UDG, to produce an abasic site and generation of single strand breaks through digestion with APE1. Normally replicated DNA generates single stranded breaks and staggered double stranded breaks in rereplicated DNA. ( B and C ) Selective fragmentation of yeast (B) and human (C) genomic DNA requires BrdU, UVA and UDG/APE-1 digestion. Genomic DNA from cells labeled with BrdU for zero or two cell cycles (−,+ BrdU) to mimic double labeled rereplicated DNA was treated with the indicated steps of the Rerep-Seq digest procedure. ( D and E ) Optimization of fragment size for yeast and human DNA. Genomic DNA exposed to varying doses of UVA followed by treatment with UGD/APE1, and DNA exposed to UVA followed by varying UDG and APE1 digestion time. ( F and G ) Selective fragmentation and enrichment of double BrdU labeled yeast DNA. ( H and I ) Selective fragmentation and enrichment of double BrdU labeled human DNA. Rerep-digest on genomic DNA treated with BrdU for 1, 2 or 3 cell cycles. DNA fragments were gel extracted and equal volumes were analyzed by qPCR with primers within ARS307 (yeast) and ACTb gene (human). Data represent the average of three biological replicates ( n = 3), error bars represent the SEM.

    Techniques Used: Synthesized, Labeling, Real-time Polymerase Chain Reaction

    Related Articles

    Concentration Assay:

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries
    Article Snippet: .. Enzyme exposure Microarrays were incubated with 1× UDG Reaction Buffer (20 mM Tris-HCl, 1 mM DTT and 1 mM EDTA pH 8) and 5 units of UDG (E. coli UDG, New England Biolabs, M0280S) in a 300 μl final volume (final enzyme concentration 0.016 U/μl) for either 1 hour or for different time periods ranging from 7 to 120 minutes (7, 15, 30, 60 and 120 min) at 37 °C in a hybridization oven (Boekel Scientific). .. Subsequently, the microarrays were rinsed in deionized water and dried in a microarray centrifuge.

    Incubation:

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries
    Article Snippet: .. Enzyme exposure Microarrays were incubated with 1× UDG Reaction Buffer (20 mM Tris-HCl, 1 mM DTT and 1 mM EDTA pH 8) and 5 units of UDG (E. coli UDG, New England Biolabs, M0280S) in a 300 μl final volume (final enzyme concentration 0.016 U/μl) for either 1 hour or for different time periods ranging from 7 to 120 minutes (7, 15, 30, 60 and 120 min) at 37 °C in a hybridization oven (Boekel Scientific). .. Subsequently, the microarrays were rinsed in deionized water and dried in a microarray centrifuge.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase
    Article Snippet: .. Treatment of FFPE DNA with uracil-DNA-glycosylase (UDG) To perform the UDG treatment and subsequent PCR/HRM assays without opening of reaction tubes, UDG (0.5 units/reaction, unless specified) and the UDG buffer (New England BioLabs, Ipswich, MA) were directly added to PCR/HRM master mixes. .. The reaction tubes were first incubated at 37°C for 30 minutes for UDG treatment, followed by the standard PCR/HRM assay conditions on the RotorGene Q instrument.

    Polymerase Chain Reaction:

    Article Title: Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism
    Article Snippet: .. The PCR product was treated with uracil DNA glycosylase (NEB) for 2 h. The abasic site was stabilized by making the solution 100 mM in freshly diluted NaBH4 and incubating on ice for 30 min. .. The DNA was then purified using a MicroSpin G-50 gel filtration device (Amersham Pharmacia).

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase
    Article Snippet: .. Treatment of FFPE DNA with uracil-DNA-glycosylase (UDG) To perform the UDG treatment and subsequent PCR/HRM assays without opening of reaction tubes, UDG (0.5 units/reaction, unless specified) and the UDG buffer (New England BioLabs, Ipswich, MA) were directly added to PCR/HRM master mixes. .. The reaction tubes were first incubated at 37°C for 30 minutes for UDG treatment, followed by the standard PCR/HRM assay conditions on the RotorGene Q instrument.

    other:

    Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity
    Article Snippet: This was then added to 10 μl of master mix containing 10 pmol Taqman probe, 0.4 units uracil DNA glycosylase, 50 mM Tris (pH 7.4), and 10 mM EDTA, and assayed as described for cell lysates.

    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).

    Article Title: The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient
    Article Snippet: Uracil DNA [Glycosylase (UDG) (M0280S), human AP endonuclease I (APE1) (M0282S), terminal transferase (M0252S), T4 PNK (M0201S)] and T4 DNA ligase (M0202S) were purchased from New England Biolabs.

    Hybridization:

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries
    Article Snippet: .. Enzyme exposure Microarrays were incubated with 1× UDG Reaction Buffer (20 mM Tris-HCl, 1 mM DTT and 1 mM EDTA pH 8) and 5 units of UDG (E. coli UDG, New England Biolabs, M0280S) in a 300 μl final volume (final enzyme concentration 0.016 U/μl) for either 1 hour or for different time periods ranging from 7 to 120 minutes (7, 15, 30, 60 and 120 min) at 37 °C in a hybridization oven (Boekel Scientific). .. Subsequently, the microarrays were rinsed in deionized water and dried in a microarray centrifuge.

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  • 99
    New England Biolabs uracil dna glycosylase
    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil <t>DNA</t> <t>glycosylase</t> (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.
    Uracil Dna Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/uracil dna glycosylase/product/New England Biolabs
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2020-07
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    91
    New England Biolabs udg
    Substrate specificity of Mj <t>UDG.</t> ( A ) Substrate specificity of Mj UDG on the 5′-end-labeled 32mer oligonucleotide substrates containing uracil (*U:A, *U:T, *U:C and *U:G) and 8-oxoG-containing 39mer substrates (*oG:A, *oG:T, *oG:C and *oG:G). After treating 1 pmol of substrate with Mj UDG for 20 min at 55°C, the reaction mixture was treated with NaOH and electrophoresed on a 15% denaturing polyacrylamide gel. The bands were visualized on a BAS 2500 image analyzer. ( B ) The average values were obtained from at least three independent experiments. ( C ) The Mj UDG reaction was carried out at 55°C with various oligonucleotide duplexes (containing uracil, *U:T; 8-oxoG, *oG:C; apurinic/apyrimidinic site, *AP:A; 3-methyladenine, *3mA:T; 7-methylguanine, *7mG:C, respectively, where the asterisk indicates the 5′-end-labeled strand). At the indicated time points, the reaction mixture was removed and treated with 100 mM NaOH at 95°C for 10 min. The cleavage products were analyzed by 15% denaturing polyacrylamide gel electrophoresis and using a BAS 2500 image analyzer. All experiments were carried out independently three times.
    Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/udg/product/New England Biolabs
    Average 91 stars, based on 45 article reviews
    Price from $9.99 to $1999.99
    udg - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

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    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

    Journal: PLoS Pathogens

    Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity

    doi: 10.1371/journal.ppat.0030135

    Figure Lengend Snippet: A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

    Article Snippet: To each well was added 10 μl of cell lysate in NP40 buffer and 70 μl of a master mix containing 10 pmol Taqman probe, 0.4 units uracil DNA glycosylase (NEB, http://www.neb.com/ ), 50 mM Tris (pH 7.4), and 10 mM EDTA.

    Techniques: Activity Assay, Transfection, Derivative Assay, Labeling, Countercurrent Chromatography, Recombinant, Incubation, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot

    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.

    Journal: Nucleic Acids Research

    Article Title: The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient

    doi: 10.1093/nar/gkm1053

    Figure Lengend Snippet: L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.

    Article Snippet: Uracil DNA [Glycosylase (UDG) (M0280S), human AP endonuclease I (APE1) (M0282S), terminal transferase (M0252S), T4 PNK (M0201S)] and T4 DNA ligase (M0202S) were purchased from New England Biolabs.

    Techniques: Purification, Incubation, Activity Assay, Primer Extension Assay

    Substrate specificity of Mj UDG. ( A ) Substrate specificity of Mj UDG on the 5′-end-labeled 32mer oligonucleotide substrates containing uracil (*U:A, *U:T, *U:C and *U:G) and 8-oxoG-containing 39mer substrates (*oG:A, *oG:T, *oG:C and *oG:G). After treating 1 pmol of substrate with Mj UDG for 20 min at 55°C, the reaction mixture was treated with NaOH and electrophoresed on a 15% denaturing polyacrylamide gel. The bands were visualized on a BAS 2500 image analyzer. ( B ) The average values were obtained from at least three independent experiments. ( C ) The Mj UDG reaction was carried out at 55°C with various oligonucleotide duplexes (containing uracil, *U:T; 8-oxoG, *oG:C; apurinic/apyrimidinic site, *AP:A; 3-methyladenine, *3mA:T; 7-methylguanine, *7mG:C, respectively, where the asterisk indicates the 5′-end-labeled strand). At the indicated time points, the reaction mixture was removed and treated with 100 mM NaOH at 95°C for 10 min. The cleavage products were analyzed by 15% denaturing polyacrylamide gel electrophoresis and using a BAS 2500 image analyzer. All experiments were carried out independently three times.

    Journal: Nucleic Acids Research

    Article Title: A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

    doi:

    Figure Lengend Snippet: Substrate specificity of Mj UDG. ( A ) Substrate specificity of Mj UDG on the 5′-end-labeled 32mer oligonucleotide substrates containing uracil (*U:A, *U:T, *U:C and *U:G) and 8-oxoG-containing 39mer substrates (*oG:A, *oG:T, *oG:C and *oG:G). After treating 1 pmol of substrate with Mj UDG for 20 min at 55°C, the reaction mixture was treated with NaOH and electrophoresed on a 15% denaturing polyacrylamide gel. The bands were visualized on a BAS 2500 image analyzer. ( B ) The average values were obtained from at least three independent experiments. ( C ) The Mj UDG reaction was carried out at 55°C with various oligonucleotide duplexes (containing uracil, *U:T; 8-oxoG, *oG:C; apurinic/apyrimidinic site, *AP:A; 3-methyladenine, *3mA:T; 7-methylguanine, *7mG:C, respectively, where the asterisk indicates the 5′-end-labeled strand). At the indicated time points, the reaction mixture was removed and treated with 100 mM NaOH at 95°C for 10 min. The cleavage products were analyzed by 15% denaturing polyacrylamide gel electrophoresis and using a BAS 2500 image analyzer. All experiments were carried out independently three times.

    Article Snippet: Restriction endonuclease, T4 DNA ligase, Escherichia coli endonuclease III (EndoIII) and UDG were purchased from New England Biolabs (MA).

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis

    Uracil-DNA glycosylase activity of Mj UDG. ( A ) Uracil-processing activity of Mj UDG on the single- or double-stranded DNA substrates. The 32mer substrates (*U-containing single strand or *U:T mismatch-containing duplex) were incubated with 5 pmol of Mj UDG at 55°C for 20 min. The reaction mixture was treated with 100 mM NaOH at 95°C for 10 min. The products were separated on a 15% denaturing polyacrylamide gel, and the bands were visualized using a BAS 2500 image analyzer. The asterisk indicates the 5′-end-labeled strand. ( B ) Comparison of uracil-DNA glycosylase activity at different temperature of Mj UDG and E.coli UDG ( Ec UDG). Mj UDG was incubated with 5′-end-labeled *U:T mismatch-containing oligonucleotide duplex at 37 or 65°C, respectively. The reaction mixture was treated with 100 mM NaOH at 95°C for 10 min. Activity assay of Ec UDG was also performed with the instruction manual. ( C ) Monofunctional activity of Mj UDG. Mj UDG protein (5 pmol) was incubated with 1 pmol of 5′-end-labeled oligonucleotide duplex (*U:T mismatch) at 55°C for 20 min. The reaction mixture was then incubated at 95°C for 10 min in the absence (lane 2) or presence (lane 3) of NaOH. ( D ) Monofunctional activity of M jUDG on 8-oxoG-containing substrate was also examined under the same conditions. After reacting 1 pmol of 8-oxoG-containing 39mer duplex (*oG:C) with Mj UDG for 20 min at 55°C, the reaction mixture was treated at 95°C for 10 min without (lane 2) or with (lane 3) NaOH.

    Journal: Nucleic Acids Research

    Article Title: A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

    doi:

    Figure Lengend Snippet: Uracil-DNA glycosylase activity of Mj UDG. ( A ) Uracil-processing activity of Mj UDG on the single- or double-stranded DNA substrates. The 32mer substrates (*U-containing single strand or *U:T mismatch-containing duplex) were incubated with 5 pmol of Mj UDG at 55°C for 20 min. The reaction mixture was treated with 100 mM NaOH at 95°C for 10 min. The products were separated on a 15% denaturing polyacrylamide gel, and the bands were visualized using a BAS 2500 image analyzer. The asterisk indicates the 5′-end-labeled strand. ( B ) Comparison of uracil-DNA glycosylase activity at different temperature of Mj UDG and E.coli UDG ( Ec UDG). Mj UDG was incubated with 5′-end-labeled *U:T mismatch-containing oligonucleotide duplex at 37 or 65°C, respectively. The reaction mixture was treated with 100 mM NaOH at 95°C for 10 min. Activity assay of Ec UDG was also performed with the instruction manual. ( C ) Monofunctional activity of Mj UDG. Mj UDG protein (5 pmol) was incubated with 1 pmol of 5′-end-labeled oligonucleotide duplex (*U:T mismatch) at 55°C for 20 min. The reaction mixture was then incubated at 95°C for 10 min in the absence (lane 2) or presence (lane 3) of NaOH. ( D ) Monofunctional activity of M jUDG on 8-oxoG-containing substrate was also examined under the same conditions. After reacting 1 pmol of 8-oxoG-containing 39mer duplex (*oG:C) with Mj UDG for 20 min at 55°C, the reaction mixture was treated at 95°C for 10 min without (lane 2) or with (lane 3) NaOH.

    Article Snippet: Restriction endonuclease, T4 DNA ligase, Escherichia coli endonuclease III (EndoIII) and UDG were purchased from New England Biolabs (MA).

    Techniques: Activity Assay, Incubation, Labeling

    Activity assay of Mj UDG protein on thymine glycol (Tg)-containing DNA substrate. Each reaction mixture was analyzed on a 15% denaturing polyacrylamide gel and the bands were visualized on a BAS 2500 image analyzer. The intact 32mer and 16mer are indicated as the substrates and products, respectively. ( A ) Different amounts of Mj UDG (5 pmol, lane 3; 20 pmol, lane 4; 100 pmol, lane 5) were incubated with 1 pmol of 5′-labeled 32mer oligonucleotide duplex containing Tg at 55°C for 20 min. The E.coli EndoIII reaction, as a control, was performed at 37°C for 20 min. ( B ) Activity assay of Mj UDG on four different substrates containing a Tg site. The reaction was carried out with duplex substrates containing four common bases opposite a Tg site at 55°C for 20 min. The asterisk indicates the 5′-end-labeled strand.

    Journal: Nucleic Acids Research

    Article Title: A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

    doi:

    Figure Lengend Snippet: Activity assay of Mj UDG protein on thymine glycol (Tg)-containing DNA substrate. Each reaction mixture was analyzed on a 15% denaturing polyacrylamide gel and the bands were visualized on a BAS 2500 image analyzer. The intact 32mer and 16mer are indicated as the substrates and products, respectively. ( A ) Different amounts of Mj UDG (5 pmol, lane 3; 20 pmol, lane 4; 100 pmol, lane 5) were incubated with 1 pmol of 5′-labeled 32mer oligonucleotide duplex containing Tg at 55°C for 20 min. The E.coli EndoIII reaction, as a control, was performed at 37°C for 20 min. ( B ) Activity assay of Mj UDG on four different substrates containing a Tg site. The reaction was carried out with duplex substrates containing four common bases opposite a Tg site at 55°C for 20 min. The asterisk indicates the 5′-end-labeled strand.

    Article Snippet: Restriction endonuclease, T4 DNA ligase, Escherichia coli endonuclease III (EndoIII) and UDG were purchased from New England Biolabs (MA).

    Techniques: Activity Assay, Incubation, Labeling

    DNA glycosylase activity of Mj UDG protein. Purified Mj UDG protein (5 pmol) was incubated with 1 pmol of 5′-end-labeled oligonucleotide duplex at 55°C for 20 min. The reaction mixture was treated with 100 mM NaOH and incubated at 95°C for 10 min. The cleavage products were analyzed by 15% denaturing PAGE and using a BAS 2500 image analyzer. The asterisk indicates the 5′-end-labeled strand. Oligonucleotide strands containing the apurinic/apyrimidinic (AP) site or 8-oxoguanine (oG) were 39mer, and strands containing adenine (A), thymine (T), thymine glycol (Tg), uracil (U), hypoxanthine (HX), 3-methyladenine (3mA) or 7-methylguanine (7mG) were 32mer. Only a reaction mixture of AP- containing substrate was not treated with NaOH and heating after reaction with Mj UDG. Each damaged base in the oligonucleotide sequences was at position 16. In the bottom graph, the quantitative data were obtained from at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

    doi:

    Figure Lengend Snippet: DNA glycosylase activity of Mj UDG protein. Purified Mj UDG protein (5 pmol) was incubated with 1 pmol of 5′-end-labeled oligonucleotide duplex at 55°C for 20 min. The reaction mixture was treated with 100 mM NaOH and incubated at 95°C for 10 min. The cleavage products were analyzed by 15% denaturing PAGE and using a BAS 2500 image analyzer. The asterisk indicates the 5′-end-labeled strand. Oligonucleotide strands containing the apurinic/apyrimidinic (AP) site or 8-oxoguanine (oG) were 39mer, and strands containing adenine (A), thymine (T), thymine glycol (Tg), uracil (U), hypoxanthine (HX), 3-methyladenine (3mA) or 7-methylguanine (7mG) were 32mer. Only a reaction mixture of AP- containing substrate was not treated with NaOH and heating after reaction with Mj UDG. Each damaged base in the oligonucleotide sequences was at position 16. In the bottom graph, the quantitative data were obtained from at least three independent experiments.

    Article Snippet: Restriction endonuclease, T4 DNA ligase, Escherichia coli endonuclease III (EndoIII) and UDG were purchased from New England Biolabs (MA).

    Techniques: Activity Assay, Purification, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

    Purification and uracil-DNA glycosylase activity of the putative UDG ( Aae UDG) from A.aeolicus . ( A ) Expression and purification of the recombinant Aae UDG protein. Lane M, protein molecular weight markers; lane 1, soluble extract of cells harboring pET28a- Aae UDG after 6 h induction with IPTG; lane 2, protein fraction eluted from an Ni-NTA affinity column; lane 3, Aae UDG protein eluted by Superdex-75 gel filtration chromatography after thrombin treatment. ( B ) Activity assay of Aae UDG on four different oligonucleotide substrates containing a uracil base (*U:A, *U:T, *U:C and *U:G). The asterisk indicates the 5′-end-labeled strand. The reaction was carried out with 32mer duplex substrates containing four common bases opposite a uracil at 55°C for 20 min. Each reaction mixture was treated with NaOH at 95°C for 10 min, and then samples were analyzed by 15% denaturing polyacrylamide gel electrophoresis and using a BAS 2500 image analyzer.

    Journal: Nucleic Acids Research

    Article Title: A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

    doi:

    Figure Lengend Snippet: Purification and uracil-DNA glycosylase activity of the putative UDG ( Aae UDG) from A.aeolicus . ( A ) Expression and purification of the recombinant Aae UDG protein. Lane M, protein molecular weight markers; lane 1, soluble extract of cells harboring pET28a- Aae UDG after 6 h induction with IPTG; lane 2, protein fraction eluted from an Ni-NTA affinity column; lane 3, Aae UDG protein eluted by Superdex-75 gel filtration chromatography after thrombin treatment. ( B ) Activity assay of Aae UDG on four different oligonucleotide substrates containing a uracil base (*U:A, *U:T, *U:C and *U:G). The asterisk indicates the 5′-end-labeled strand. The reaction was carried out with 32mer duplex substrates containing four common bases opposite a uracil at 55°C for 20 min. Each reaction mixture was treated with NaOH at 95°C for 10 min, and then samples were analyzed by 15% denaturing polyacrylamide gel electrophoresis and using a BAS 2500 image analyzer.

    Article Snippet: Restriction endonuclease, T4 DNA ligase, Escherichia coli endonuclease III (EndoIII) and UDG were purchased from New England Biolabs (MA).

    Techniques: Purification, Activity Assay, Expressing, Recombinant, Molecular Weight, Affinity Column, Filtration, Chromatography, Labeling, Polyacrylamide Gel Electrophoresis