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  • 99
    New England Biolabs uracil dna n 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 N Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher uracil dna n glycosylase
    (A) Targeted disruption of mNth1. The mNth1 locus consists of five exons, as represented by the numbered black boxes in the schematic of the wild-type allele. To generate the recombinant allele, exons 2 through 4 were replaced with the neomycin resistance cassette via homologous recombination. The positions of the PCR primers used in routine genotyping are indicated by arrows, and their sequences are numbered based upon their location in the mNth1 cDNA. Abbreviations: B, Bam HI; Bg, Bgl II; E, Eco RI; K, Kpn I; RV, Eco RV; S, Sac I; Sp, Spe I; X, Xba I;Xh, Xho I. (B) Southern blot analysis of ES cell clones. In the left panel, probing of the 5′ region after Eco RI and Eco RV digestion of genomic DNA yielded a 7.4-kb band for the wild-type allele and a 4.5-kb band for the recombinant allele, due to the insertion of a new Eco RV (RV) site in the targeting vector. In the right panel, probing of the 3′ region after Spe I digestion of genomic DNA produced an 18.0-kb band for the wild-type allele and a 14.4-kb band for the recombinant allele, due to the insertion of a new Spe I (Sp) site in the targeting vector. (C) mNth1 gene dosage effect in ES cells. The enzymatic activity of parental mNth1 +/+ (wild type [WT]) and candidate heterozygous (HT) mNth1 +/− clones (clone 73 here) were analyzed by <t>DNA</t> N <t>-glycosylase</t> base release assay. ES cells were lysed, and 200 or 400 μg of cellular protein was incubated with 100 ng of UV-irradiated poly(dG-[ 3 H]dC) for 2 or 4 h. Base release was measured by liquid scintillation counting. The results of this experiment are representative of the results obtained with all mNth1 +/− clones tested.
    Uracil Dna N Glycosylase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher uracil dna glycosylase
    ( Left ) Denaturing PAGE (20% gels) of 3′-end-labeled fragments obtained from <t>uracil-DNA</t> <t>glycosylase</t> (UDG) activity on a 25-mer oligonucleotide containing dU at the same position as used for pBQ-dC. Lane 2 shows that after UDG treatment the fragment migrates with p17-mer (lane 4) and not with the 17-mer (lane 3). The same procedures for identification of the pBQ-dC endonuclease-treated oligomer (lane 8) did not produce the 17-mer, p17-mer, the 18-mer (lane 5), or p18-mer (lanes 6 and 9). ( Right ) Size and charge markers to identify the 3′ cleavage product of the pBQ-dC oligomer are in lanes 4–6. Only a synthetic pBQ-dC-18-mer (lane 4) coincided in migration mobility with the pBQ-dC endonuclease-produced 3′ fragment after dephosphorylation (lane 3). This gel shows that it is possible to distinguish between phosphorylated (lane 2) and dephosphorylated (lane 3) cleavage products.
    Uracil Dna Glycosylase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore uracil dna glycosylase udg
    Generation and validation of the splice acceptor plasmid library. ( A ) Schematics showing the split-GFP trans -splicing screening approach. LV derived splice donor expressing N-terminal GFP and full Synapsin I intron 9–10 and splice acceptor expressing intron fragment, C-terminal GFP and <t>DNA</t> barcode (BC). The trans -spliced mRNA is a hybrid between donor and acceptor and the result is the full open reading frame of GFP. By deep sequencing of molecular barcodes and intron fragments from the plasmid library prior to the cell culture or in vivo screening assay, a look-up table can be created to create a link between the two. ( B ) A schematic overview of the process used for the preparation of intron fragments for library cloning by using dUTP-based fragmentation via Uracil DNA <t>Glycosylase</t> and NaOH followed by end-repair and A-tailing. ( C ) Plot of the overall coverage of the Synapsin I intron with the negative (active) strand in red and positive (inactive) strand orientation in blue. ( D for additional information.)
    Uracil Dna Glycosylase Udg, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Meridian Life Science uracil dna glycoylase
    Generation and validation of the splice acceptor plasmid library. ( A ) Schematics showing the split-GFP trans -splicing screening approach. LV derived splice donor expressing N-terminal GFP and full Synapsin I intron 9–10 and splice acceptor expressing intron fragment, C-terminal GFP and <t>DNA</t> barcode (BC). The trans -spliced mRNA is a hybrid between donor and acceptor and the result is the full open reading frame of GFP. By deep sequencing of molecular barcodes and intron fragments from the plasmid library prior to the cell culture or in vivo screening assay, a look-up table can be created to create a link between the two. ( B ) A schematic overview of the process used for the preparation of intron fragments for library cloning by using dUTP-based fragmentation via Uracil DNA <t>Glycosylase</t> and NaOH followed by end-repair and A-tailing. ( C ) Plot of the overall coverage of the Synapsin I intron with the negative (active) strand in red and positive (inactive) strand orientation in blue. ( D for additional information.)
    Uracil Dna Glycoylase, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    (A) Targeted disruption of mNth1. The mNth1 locus consists of five exons, as represented by the numbered black boxes in the schematic of the wild-type allele. To generate the recombinant allele, exons 2 through 4 were replaced with the neomycin resistance cassette via homologous recombination. The positions of the PCR primers used in routine genotyping are indicated by arrows, and their sequences are numbered based upon their location in the mNth1 cDNA. Abbreviations: B, Bam HI; Bg, Bgl II; E, Eco RI; K, Kpn I; RV, Eco RV; S, Sac I; Sp, Spe I; X, Xba I;Xh, Xho I. (B) Southern blot analysis of ES cell clones. In the left panel, probing of the 5′ region after Eco RI and Eco RV digestion of genomic DNA yielded a 7.4-kb band for the wild-type allele and a 4.5-kb band for the recombinant allele, due to the insertion of a new Eco RV (RV) site in the targeting vector. In the right panel, probing of the 3′ region after Spe I digestion of genomic DNA produced an 18.0-kb band for the wild-type allele and a 14.4-kb band for the recombinant allele, due to the insertion of a new Spe I (Sp) site in the targeting vector. (C) mNth1 gene dosage effect in ES cells. The enzymatic activity of parental mNth1 +/+ (wild type [WT]) and candidate heterozygous (HT) mNth1 +/− clones (clone 73 here) were analyzed by DNA N -glycosylase base release assay. ES cells were lysed, and 200 or 400 μg of cellular protein was incubated with 100 ng of UV-irradiated poly(dG-[ 3 H]dC) for 2 or 4 h. Base release was measured by liquid scintillation counting. The results of this experiment are representative of the results obtained with all mNth1 +/− clones tested.

    Journal: Molecular and Cellular Biology

    Article Title: Targeted Deletion of mNth1 Reveals a Novel DNA Repair Enzyme Activity

    doi: 10.1128/MCB.22.17.6111-6121.2002

    Figure Lengend Snippet: (A) Targeted disruption of mNth1. The mNth1 locus consists of five exons, as represented by the numbered black boxes in the schematic of the wild-type allele. To generate the recombinant allele, exons 2 through 4 were replaced with the neomycin resistance cassette via homologous recombination. The positions of the PCR primers used in routine genotyping are indicated by arrows, and their sequences are numbered based upon their location in the mNth1 cDNA. Abbreviations: B, Bam HI; Bg, Bgl II; E, Eco RI; K, Kpn I; RV, Eco RV; S, Sac I; Sp, Spe I; X, Xba I;Xh, Xho I. (B) Southern blot analysis of ES cell clones. In the left panel, probing of the 5′ region after Eco RI and Eco RV digestion of genomic DNA yielded a 7.4-kb band for the wild-type allele and a 4.5-kb band for the recombinant allele, due to the insertion of a new Eco RV (RV) site in the targeting vector. In the right panel, probing of the 3′ region after Spe I digestion of genomic DNA produced an 18.0-kb band for the wild-type allele and a 14.4-kb band for the recombinant allele, due to the insertion of a new Spe I (Sp) site in the targeting vector. (C) mNth1 gene dosage effect in ES cells. The enzymatic activity of parental mNth1 +/+ (wild type [WT]) and candidate heterozygous (HT) mNth1 +/− clones (clone 73 here) were analyzed by DNA N -glycosylase base release assay. ES cells were lysed, and 200 or 400 μg of cellular protein was incubated with 100 ng of UV-irradiated poly(dG-[ 3 H]dC) for 2 or 4 h. Base release was measured by liquid scintillation counting. The results of this experiment are representative of the results obtained with all mNth1 +/− clones tested.

    Article Snippet: The AP site was made by treatment with uracil DNA N- glycosylase (Life Technologies) (1 U/μl).

    Techniques: Recombinant, Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Clone Assay, Plasmid Preparation, Produced, Activity Assay, Release Assay, Incubation, Irradiation

    Differential processing of Tg:A (circles) versus Tg:G (squares) by hNth1 (20 nM). Solid symbols denote AP lyase activity (neutral pH). Open symbols denote combined DNA N -glycosylase/AP lyase activity of hNth1 against the substrate (putrescine, alkaline pH). Reaction time was 30 min.

    Journal: Molecular and Cellular Biology

    Article Title: Targeted Deletion of mNth1 Reveals a Novel DNA Repair Enzyme Activity

    doi: 10.1128/MCB.22.17.6111-6121.2002

    Figure Lengend Snippet: Differential processing of Tg:A (circles) versus Tg:G (squares) by hNth1 (20 nM). Solid symbols denote AP lyase activity (neutral pH). Open symbols denote combined DNA N -glycosylase/AP lyase activity of hNth1 against the substrate (putrescine, alkaline pH). Reaction time was 30 min.

    Article Snippet: The AP site was made by treatment with uracil DNA N- glycosylase (Life Technologies) (1 U/μl).

    Techniques: Activity Assay

    Effects of differential folate repletion on inherent DNA damage and uracil misincorporation in 10 day folate depleted HaCaT cells as measured by comet assay with (solid bars) or without (open bars) UDG treatment. Cells cultured for 10 days in folate free

    Journal: Journal of photochemistry and photobiology. B, Biology

    Article Title: Photobiological Implications of Folate Depletion and Repletion in Cultured Human Keratinocytes

    doi: 10.1016/j.jphotobiol.2010.02.003

    Figure Lengend Snippet: Effects of differential folate repletion on inherent DNA damage and uracil misincorporation in 10 day folate depleted HaCaT cells as measured by comet assay with (solid bars) or without (open bars) UDG treatment. Cells cultured for 10 days in folate free

    Article Snippet: Briefly, after cell lysis, the slides were washed three times for 5 min each in uracil DNA glycosylase (UDG) buffer (60 mM Tris-HCl, 1 mM EDTA, 0.1 mg/mL BSA, pH 8.0) (Fermentas).

    Techniques: Single Cell Gel Electrophoresis, Cell Culture

    Effects of folate restriction on inherent DNA damage and uracil misincorporation as measured by comet assay with (solid bars) or without (open bars) uracil DNA glycosylase (UDG) treatment. Results are mean ± SEM. *** P

    Journal: Journal of photochemistry and photobiology. B, Biology

    Article Title: Photobiological Implications of Folate Depletion and Repletion in Cultured Human Keratinocytes

    doi: 10.1016/j.jphotobiol.2010.02.003

    Figure Lengend Snippet: Effects of folate restriction on inherent DNA damage and uracil misincorporation as measured by comet assay with (solid bars) or without (open bars) uracil DNA glycosylase (UDG) treatment. Results are mean ± SEM. *** P

    Article Snippet: Briefly, after cell lysis, the slides were washed three times for 5 min each in uracil DNA glycosylase (UDG) buffer (60 mM Tris-HCl, 1 mM EDTA, 0.1 mg/mL BSA, pH 8.0) (Fermentas).

    Techniques: Single Cell Gel Electrophoresis

    Dnmt3a stimulates the glycosylase activity of TDG. ( A ) Purified recombinant 6xHis-tagged Dnmt3a, APE and TDG proteins (Coomassie blue staining). The purity of Dnmt3a, APE and TDG was about 85, 90 and 80%, respectively, as determined by densitometry. Lower panel, the G/T mismatch-containing substrate (27 bp) used for the glycosylase activity assay. The upper T-containing strand was labeled with 32 P at the 5′ end. ( B ) Glycosylase activity assay for TDG in the absence and presence of Dnmt3a or APE. The reactions in lanes 1–9 contained 25 mM HEPES (pH 7.8), 0.5 mM EDTA, 0.5 mM DTT, 0.5 mg/ml BSA and 10 nM substrate DNA. The reactions in lanes 10–12 contained 50 mM NaCl and 2 mM MgCl 2 in addition to the above and show no difference, ruling out any possibility that the stimulation is only due to the addition of salt from the Dnmt3a prep. All reactions were incubated at 30°C for 1 h. The excision of unpaired thymine resulted in breakage of the upper strand upon subsequent hot alkaline treatment. The generation of the 13mer breakage product was examined by separation on a denaturing gel followed by quantification with phosphoimaging. The lower panel is a bar graph representation of TDG enzymatic assays. The activity of TDG alone was set to 1. The relative activity (given on the y -axis) for each reaction is the average of three experiments ±SD. ( C ) The kinetics of the base-excision reaction of TDG with and without Dnmt3a. DNA substrate (10 nM) was incubated with TDG (4 nM) alone or with Dnmt3a (20 nM) in the reaction buffer without NaCl for different time periods indicated on the top. The lower panel is a line graph representation in which the activity for each reaction at different time points is the average of three experiments ±SD. ( D ) Specific stimulation of TDG glycosylase activity by Dnmt3a. The glycosylase reaction was carried out for 30 min at 30°C. The upper panel shows the lack of stimulation of two other glycosylases UDG and SMUG1 by Dnmt3a. The DNA substrate used here had the same sequence as shown in (A) except that a G/U mismatch was incorporated in the place of G/T. The lower panel shows the absence of stimulation by M.SssI, M.HhaI and Dnmt2 on the glycosylase activity of TDG.

    Journal: Nucleic Acids Research

    Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair

    doi: 10.1093/nar/gkl1052

    Figure Lengend Snippet: Dnmt3a stimulates the glycosylase activity of TDG. ( A ) Purified recombinant 6xHis-tagged Dnmt3a, APE and TDG proteins (Coomassie blue staining). The purity of Dnmt3a, APE and TDG was about 85, 90 and 80%, respectively, as determined by densitometry. Lower panel, the G/T mismatch-containing substrate (27 bp) used for the glycosylase activity assay. The upper T-containing strand was labeled with 32 P at the 5′ end. ( B ) Glycosylase activity assay for TDG in the absence and presence of Dnmt3a or APE. The reactions in lanes 1–9 contained 25 mM HEPES (pH 7.8), 0.5 mM EDTA, 0.5 mM DTT, 0.5 mg/ml BSA and 10 nM substrate DNA. The reactions in lanes 10–12 contained 50 mM NaCl and 2 mM MgCl 2 in addition to the above and show no difference, ruling out any possibility that the stimulation is only due to the addition of salt from the Dnmt3a prep. All reactions were incubated at 30°C for 1 h. The excision of unpaired thymine resulted in breakage of the upper strand upon subsequent hot alkaline treatment. The generation of the 13mer breakage product was examined by separation on a denaturing gel followed by quantification with phosphoimaging. The lower panel is a bar graph representation of TDG enzymatic assays. The activity of TDG alone was set to 1. The relative activity (given on the y -axis) for each reaction is the average of three experiments ±SD. ( C ) The kinetics of the base-excision reaction of TDG with and without Dnmt3a. DNA substrate (10 nM) was incubated with TDG (4 nM) alone or with Dnmt3a (20 nM) in the reaction buffer without NaCl for different time periods indicated on the top. The lower panel is a line graph representation in which the activity for each reaction at different time points is the average of three experiments ±SD. ( D ) Specific stimulation of TDG glycosylase activity by Dnmt3a. The glycosylase reaction was carried out for 30 min at 30°C. The upper panel shows the lack of stimulation of two other glycosylases UDG and SMUG1 by Dnmt3a. The DNA substrate used here had the same sequence as shown in (A) except that a G/U mismatch was incorporated in the place of G/T. The lower panel shows the absence of stimulation by M.SssI, M.HhaI and Dnmt2 on the glycosylase activity of TDG.

    Article Snippet: Uracil DNA glycosylase (UDG) and micrococcal nuclease were from Fermentas.

    Techniques: Activity Assay, Purification, Recombinant, Staining, Labeling, Incubation, Sequencing

    Effects of EDTA on MutY Dr binding and glycosylase activities with A/G- and A/GO-containing DNA. (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).

    Journal: Journal of Bacteriology

    Article Title: Molecular Cloning and Functional Analysis of the MutY Homolog of Deinococcus radiodurans

    doi: 10.1128/JB.183.21.6151-6158.2001

    Figure Lengend Snippet: Effects of EDTA on MutY Dr binding and glycosylase activities with A/G- and A/GO-containing DNA. (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).

    Article Snippet: DNA substrates containing U/G or U/GO (300 fmol) were fully converted to AP/G- or AP/GO-containing DNA substrates by treatment with 1.5 U of E. coli uracil DNA glycosylase (Life Technologies, Inc.) at 37°C for 1 h in a buffer containing 20 mM Tris-HCl (pH 7.6), 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 2.9% glycerol.

    Techniques: Binding Assay, Activity Assay, DNA Sequencing

    Glycosylase activities of MutY Dr (DrMutY) and MutY Ec (EcMutY) with different mismatches. Oligonucleotide substrates (3′-end-labeled 20-mers; 1.8 fmol) containing the mismatches indicated above the lanes were incubated with 3.6 nM MutY Dr (lanes 1 to 8) or 3.6 nM MutY Ec (lanes 9 to 16) for 30 min at 37°C. Abbreviations: O, GO; 2, 2-aminopurine. The arrows indicate the positions of intact DNA substrate (arrow I) and the cleaved DNA fragment (arrow N). The minor band above the cleaved product was derived from an impurity of DNA substrates that appeared above the intact DNA.

    Journal: Journal of Bacteriology

    Article Title: Molecular Cloning and Functional Analysis of the MutY Homolog of Deinococcus radiodurans

    doi: 10.1128/JB.183.21.6151-6158.2001

    Figure Lengend Snippet: Glycosylase activities of MutY Dr (DrMutY) and MutY Ec (EcMutY) with different mismatches. Oligonucleotide substrates (3′-end-labeled 20-mers; 1.8 fmol) containing the mismatches indicated above the lanes were incubated with 3.6 nM MutY Dr (lanes 1 to 8) or 3.6 nM MutY Ec (lanes 9 to 16) for 30 min at 37°C. Abbreviations: O, GO; 2, 2-aminopurine. The arrows indicate the positions of intact DNA substrate (arrow I) and the cleaved DNA fragment (arrow N). The minor band above the cleaved product was derived from an impurity of DNA substrates that appeared above the intact DNA.

    Article Snippet: DNA substrates containing U/G or U/GO (300 fmol) were fully converted to AP/G- or AP/GO-containing DNA substrates by treatment with 1.5 U of E. coli uracil DNA glycosylase (Life Technologies, Inc.) at 37°C for 1 h in a buffer containing 20 mM Tris-HCl (pH 7.6), 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 2.9% glycerol.

    Techniques: Labeling, Incubation, Derivative Assay

    ( Left ) Denaturing PAGE (20% gels) of 3′-end-labeled fragments obtained from uracil-DNA glycosylase (UDG) activity on a 25-mer oligonucleotide containing dU at the same position as used for pBQ-dC. Lane 2 shows that after UDG treatment the fragment migrates with p17-mer (lane 4) and not with the 17-mer (lane 3). The same procedures for identification of the pBQ-dC endonuclease-treated oligomer (lane 8) did not produce the 17-mer, p17-mer, the 18-mer (lane 5), or p18-mer (lanes 6 and 9). ( Right ) Size and charge markers to identify the 3′ cleavage product of the pBQ-dC oligomer are in lanes 4–6. Only a synthetic pBQ-dC-18-mer (lane 4) coincided in migration mobility with the pBQ-dC endonuclease-produced 3′ fragment after dephosphorylation (lane 3). This gel shows that it is possible to distinguish between phosphorylated (lane 2) and dephosphorylated (lane 3) cleavage products.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: An unusual mechanism for the major human apurinic/apyrimidinic (AP) endonuclease involving 5? cleavage of DNA containing a benzene-derived exocyclic adduct in the absence of an AP site

    doi:

    Figure Lengend Snippet: ( Left ) Denaturing PAGE (20% gels) of 3′-end-labeled fragments obtained from uracil-DNA glycosylase (UDG) activity on a 25-mer oligonucleotide containing dU at the same position as used for pBQ-dC. Lane 2 shows that after UDG treatment the fragment migrates with p17-mer (lane 4) and not with the 17-mer (lane 3). The same procedures for identification of the pBQ-dC endonuclease-treated oligomer (lane 8) did not produce the 17-mer, p17-mer, the 18-mer (lane 5), or p18-mer (lanes 6 and 9). ( Right ) Size and charge markers to identify the 3′ cleavage product of the pBQ-dC oligomer are in lanes 4–6. Only a synthetic pBQ-dC-18-mer (lane 4) coincided in migration mobility with the pBQ-dC endonuclease-produced 3′ fragment after dephosphorylation (lane 3). This gel shows that it is possible to distinguish between phosphorylated (lane 2) and dephosphorylated (lane 3) cleavage products.

    Article Snippet: In contrast, when a 25-mer with dU placed at the same position as pBQ-dC was used as substrate for uracil DNA glycosylase (UDG, GIBCO/BRL), the electrophoretic mobility of the cleavage fragment (lane 2) is identical to that of the phosphorylated 17-mer (lane 4).

    Techniques: Polyacrylamide Gel Electrophoresis, Labeling, Activity Assay, Migration, Produced, De-Phosphorylation Assay

    Generation and validation of the splice acceptor plasmid library. ( A ) Schematics showing the split-GFP trans -splicing screening approach. LV derived splice donor expressing N-terminal GFP and full Synapsin I intron 9–10 and splice acceptor expressing intron fragment, C-terminal GFP and DNA barcode (BC). The trans -spliced mRNA is a hybrid between donor and acceptor and the result is the full open reading frame of GFP. By deep sequencing of molecular barcodes and intron fragments from the plasmid library prior to the cell culture or in vivo screening assay, a look-up table can be created to create a link between the two. ( B ) A schematic overview of the process used for the preparation of intron fragments for library cloning by using dUTP-based fragmentation via Uracil DNA Glycosylase and NaOH followed by end-repair and A-tailing. ( C ) Plot of the overall coverage of the Synapsin I intron with the negative (active) strand in red and positive (inactive) strand orientation in blue. ( D for additional information.)

    Journal: RNA

    Article Title: Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing

    doi: 10.1261/rna.063925.117

    Figure Lengend Snippet: Generation and validation of the splice acceptor plasmid library. ( A ) Schematics showing the split-GFP trans -splicing screening approach. LV derived splice donor expressing N-terminal GFP and full Synapsin I intron 9–10 and splice acceptor expressing intron fragment, C-terminal GFP and DNA barcode (BC). The trans -spliced mRNA is a hybrid between donor and acceptor and the result is the full open reading frame of GFP. By deep sequencing of molecular barcodes and intron fragments from the plasmid library prior to the cell culture or in vivo screening assay, a look-up table can be created to create a link between the two. ( B ) A schematic overview of the process used for the preparation of intron fragments for library cloning by using dUTP-based fragmentation via Uracil DNA Glycosylase and NaOH followed by end-repair and A-tailing. ( C ) Plot of the overall coverage of the Synapsin I intron with the negative (active) strand in red and positive (inactive) strand orientation in blue. ( D for additional information.)

    Article Snippet: Intron 9–10 with dUTPs incorporated was then fragmented by using Uracil-DNA-Glycosylase (UDG) (Sigma-Aldrich) followed by NaOH ( ).

    Techniques: Plasmid Preparation, Derivative Assay, Expressing, Sequencing, Cell Culture, In Vivo, Screening Assay, Clone Assay

    APOBEC1 mediated nuclear DNA editing and damage. a Graphical representation of nuclear DNA editing by A1 proteins. The last positive 3DPCR bands retrieved bands by CMYC specific 3DPCR amplification are represented on the gradient. b Selection of hypermutated CMYC sequences after mouse A1-UGI transfection in QT6 cells for PCR products retrieved at 89.4 °C. c Dinucleotide analysis of mouse A1 deamination context performed on nuclear DNA for PCR products retrieved at 89.4 °C. Dinucleotide context expected values, based on the dinucleotide composition of DNA sequences are represented by white histograms. * Significant deviation from expected values (χ 2 -test, P

    Journal: BMC Genomics

    Article Title: Mouse APOBEC1 cytidine deaminase can induce somatic mutations in chromosomal DNA

    doi: 10.1186/s12864-019-6216-x

    Figure Lengend Snippet: APOBEC1 mediated nuclear DNA editing and damage. a Graphical representation of nuclear DNA editing by A1 proteins. The last positive 3DPCR bands retrieved bands by CMYC specific 3DPCR amplification are represented on the gradient. b Selection of hypermutated CMYC sequences after mouse A1-UGI transfection in QT6 cells for PCR products retrieved at 89.4 °C. c Dinucleotide analysis of mouse A1 deamination context performed on nuclear DNA for PCR products retrieved at 89.4 °C. Dinucleotide context expected values, based on the dinucleotide composition of DNA sequences are represented by white histograms. * Significant deviation from expected values (χ 2 -test, P

    Article Snippet: Dual promoter vector encoding uracil-DNA glycosylase inhibitor UGI from Bacillus subtilis phage, was generated using BamHI/NheI restriction sites to substitute PGK driven GFP sequence from pSF-CMV-PGK-daGFP vector (Sigma) by UGI sequence cloned into pcDNA3.1 vector.

    Techniques: Amplification, Selection, Transfection, Polymerase Chain Reaction