uracil dna glycosylase  (New England Biolabs)


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    Name:
    Uracil DNA Glycosylase
    Description:

    Catalog Number:
    M0280
    Price:
    307
    Category:
    DNA Modifying Enzymes
    Applications:
    DNA Manipulation
    Size:
    5000 units
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    Structured Review

    New England Biolabs uracil dna glycosylase
    Uracil DNA Glycosylase

    https://www.bioz.com/result/uracil dna glycosylase/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2021-07
    98/100 stars

    Images

    1) Product Images from "T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

    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.
    Figure Legend 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.

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

    2) Product Images from "Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair"

    Article Title: Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair

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

    doi: 10.1073/pnas.042700399

    Preparation of site-specific DNA–protein crosslinks. ( A ) Sequence of the uracil-containing 60-mer oligodeoxynucleotide. ( B ) Urea-PAGE showing DNA substrate preparation. Lane 1, uracil-containing 60-mer; lane 2, uracil-containing 60-mer, digested with uracil DNA glycosylase and tested with T4-pdg (control of AP-site formation); reduced AP site-containing DNA before (lane 3) and after (lanes 4–6) purification; DPC-containing DNA before (lane 7) and after (lanes 8–10) purification. After purification, DNAs were subjected to the restriction endonuclease digestion with Sna ) or Hae ). ( C ) SDS/PAGE showing DPC-containing DNA substrates before (lane 1) and after (lane 2) Hae III digestion.
    Figure Legend Snippet: Preparation of site-specific DNA–protein crosslinks. ( A ) Sequence of the uracil-containing 60-mer oligodeoxynucleotide. ( B ) Urea-PAGE showing DNA substrate preparation. Lane 1, uracil-containing 60-mer; lane 2, uracil-containing 60-mer, digested with uracil DNA glycosylase and tested with T4-pdg (control of AP-site formation); reduced AP site-containing DNA before (lane 3) and after (lanes 4–6) purification; DPC-containing DNA before (lane 7) and after (lanes 8–10) purification. After purification, DNAs were subjected to the restriction endonuclease digestion with Sna ) or Hae ). ( C ) SDS/PAGE showing DPC-containing DNA substrates before (lane 1) and after (lane 2) Hae III digestion.

    Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Purification, SDS Page

    3) Product Images from "Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases"

    Article Title: Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2013.09.004

    Role of the void-filling Met72 and adjacent His73 in lesion excision. Glycosylase assays with double-stranded Sp1:C (A) and ssSp1 (B) where the DNA substrate (20 nM) was combined with 16 nM of either WT or mutant MvNei2. WT MvNei2 is displayed as circles.
    Figure Legend Snippet: Role of the void-filling Met72 and adjacent His73 in lesion excision. Glycosylase assays with double-stranded Sp1:C (A) and ssSp1 (B) where the DNA substrate (20 nM) was combined with 16 nM of either WT or mutant MvNei2. WT MvNei2 is displayed as circles.

    Techniques Used: Mutagenesis

    4) Product Images from "Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA"

    Article Title: Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

    Journal: Nucleic Acids Research

    doi:

    8-OxoG DNA glycosylase activity of hFPG1. ( A ) An aliquot of 30 ng of purified E.coli Apn1, Fpg, hOGG1, hFPG1 or no enzyme was incubated with 100 fmol of a 24 bp duplex oligodeoxyribonucleotide containing a single 8-oxoG residue opposite A, C, G or T. Strand cleavage was analysed by 20% PAGE and phosphorimaging. ( B ) Quantification of the strand cleavage reactions. Results represent the averages of three independent experiments and error bars indicate standard deviation.
    Figure Legend Snippet: 8-OxoG DNA glycosylase activity of hFPG1. ( A ) An aliquot of 30 ng of purified E.coli Apn1, Fpg, hOGG1, hFPG1 or no enzyme was incubated with 100 fmol of a 24 bp duplex oligodeoxyribonucleotide containing a single 8-oxoG residue opposite A, C, G or T. Strand cleavage was analysed by 20% PAGE and phosphorimaging. ( B ) Quantification of the strand cleavage reactions. Results represent the averages of three independent experiments and error bars indicate standard deviation.

    Techniques Used: Activity Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis, Standard Deviation

    FaPy DNA glycosylase activity of hFPG1 and hFPG2. ( A ) Different amounts of cell extracts from uninfected and baculovirus-infected insect cells expressing APE2, hFPG2 or hFPG1 from appropriate cDNA constructs were assayed for removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC) (0.4 µg). Diamonds, hFPG1; small squares, hFPG2; triangles, APE2; large squares, uninfected. ( B ) Removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC) DNA by increasing amounts of purified E.coli Fpg (triangles), hOGG1 (squares) and hFPG1 (diamonds).
    Figure Legend Snippet: FaPy DNA glycosylase activity of hFPG1 and hFPG2. ( A ) Different amounts of cell extracts from uninfected and baculovirus-infected insect cells expressing APE2, hFPG2 or hFPG1 from appropriate cDNA constructs were assayed for removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC) (0.4 µg). Diamonds, hFPG1; small squares, hFPG2; triangles, APE2; large squares, uninfected. ( B ) Removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC) DNA by increasing amounts of purified E.coli Fpg (triangles), hOGG1 (squares) and hFPG1 (diamonds).

    Techniques Used: Activity Assay, Infection, Expressing, Construct, Purification

    5-ohC DNA glycosylase activity of hFPG1. ( A ) Incision and ( B ) probing for covalent hFPG1 DNA intermediates by NaCNBH 3 of 5-ohC-containing DNA by hFPG1. An aliquot of 30 ng of purified E.coli Nth, Nei, Fpg, hOGG1, hFPG1 or no enzyme was incubated with 100 fmol of a 40 bp duplex oligodeoxyribonucleotide containing a single 5-ohC residue opposite G. Strand cleavage was analysed by 20% denaturing PAGE and bands detected by phosphorimaging. Protein–DNA complexes were separated by 10% Tricine–SDS–PAGE and detected by phosphorimaging. ( C ) Increasing amounts of purified E.coli Nei (diamonds), E.coli Fpg (triangles) and hFPG1 (squares) were incubated with 100 fmol of a 40 bp duplex oligodeoxyribonucleotide containing a single 5-ohC residue opposite G, and strand cleavage was quantified by 20% PAGE followed by phosphorimaging.
    Figure Legend Snippet: 5-ohC DNA glycosylase activity of hFPG1. ( A ) Incision and ( B ) probing for covalent hFPG1 DNA intermediates by NaCNBH 3 of 5-ohC-containing DNA by hFPG1. An aliquot of 30 ng of purified E.coli Nth, Nei, Fpg, hOGG1, hFPG1 or no enzyme was incubated with 100 fmol of a 40 bp duplex oligodeoxyribonucleotide containing a single 5-ohC residue opposite G. Strand cleavage was analysed by 20% denaturing PAGE and bands detected by phosphorimaging. Protein–DNA complexes were separated by 10% Tricine–SDS–PAGE and detected by phosphorimaging. ( C ) Increasing amounts of purified E.coli Nei (diamonds), E.coli Fpg (triangles) and hFPG1 (squares) were incubated with 100 fmol of a 40 bp duplex oligodeoxyribonucleotide containing a single 5-ohC residue opposite G, and strand cleavage was quantified by 20% PAGE followed by phosphorimaging.

    Techniques Used: Activity Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page

    5) Product Images from "APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A"

    Article Title: APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1201

    Characterization of A1 RNA binding and ssDNA deamination activity. ( A ) Determination of nucleic acids that co-purify with A1. Experimental outline (sketch) shown alongside visualization of samples after denaturing gel electrophoresis. ( B ) Determination of the preferential deamination motif of A1 using the Uracil DNA Glycosylase (UDG) assay (sketch, left). Deamination of an 85 nt ssDNA with deaminated cytosines spaced 30 nt apart and an internal fluorescein label (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of 67 and 48 nt fragments, respectively; double deamination of both residues results in a 30 nt fragment. The 85 nt ssDNA substrates contained different deamination motifs, as labeled above the gel, to determine the optimal substrate. The specific activity (S.A.) was measured as pmol substrate deaminated/μg enzyme/min (pmol/μg/min) ( C ) Deamination of a 118 nt ssDNA substrate with deamination motifs spaced 63 nt apart and an internal fluorescein level (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of labeled 110 and 81 nt fragments, respectively; double deamination of both C residues on the same ssDNA results in a 63 nt fragment. A1 was either treated or not treated with RNase A. Analysis to determine a processivity factor (P.F.) is illustrated in the sketch, left. (B and C) The measurements of S.A., P.F., or standard deviation (S.D.) from three independent experiments are indicated below the gel.
    Figure Legend Snippet: Characterization of A1 RNA binding and ssDNA deamination activity. ( A ) Determination of nucleic acids that co-purify with A1. Experimental outline (sketch) shown alongside visualization of samples after denaturing gel electrophoresis. ( B ) Determination of the preferential deamination motif of A1 using the Uracil DNA Glycosylase (UDG) assay (sketch, left). Deamination of an 85 nt ssDNA with deaminated cytosines spaced 30 nt apart and an internal fluorescein label (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of 67 and 48 nt fragments, respectively; double deamination of both residues results in a 30 nt fragment. The 85 nt ssDNA substrates contained different deamination motifs, as labeled above the gel, to determine the optimal substrate. The specific activity (S.A.) was measured as pmol substrate deaminated/μg enzyme/min (pmol/μg/min) ( C ) Deamination of a 118 nt ssDNA substrate with deamination motifs spaced 63 nt apart and an internal fluorescein level (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of labeled 110 and 81 nt fragments, respectively; double deamination of both C residues on the same ssDNA results in a 63 nt fragment. A1 was either treated or not treated with RNase A. Analysis to determine a processivity factor (P.F.) is illustrated in the sketch, left. (B and C) The measurements of S.A., P.F., or standard deviation (S.D.) from three independent experiments are indicated below the gel.

    Techniques Used: RNA Binding Assay, Activity Assay, Nucleic Acid Electrophoresis, Labeling, Standard Deviation

    Effect of A1 expression on γH2AX foci. Cancerous lung cell line NCI-H1563 with or without stable doxycycline (dox) inducible A1-Flag was subjected to different conditions before staining with Flag and γH2AX antibodies. Nuclei were stained with DAPI. The NCI-H1563 were not transduced to express A1 and exposed to dox (Mock), transduced to express A1 and exposed to dox and uracil DNA glycosylase inhibitor (Induced+UGI), transduced to express A1 and not exposed to dox (Uninduced), and transduced to express A1 and exposed to dox (Induced). These conditions were carried out after ( A and B ) 24 h and ( C and D ) 48 h. (B andD) Histogram shows the numbers of foci/cell in bins of 1–5 (5), 6–10 (10), 11–15 (15) and 16 or more (More). The experiment was independently repeated in triplicate. Representative images are shown.
    Figure Legend Snippet: Effect of A1 expression on γH2AX foci. Cancerous lung cell line NCI-H1563 with or without stable doxycycline (dox) inducible A1-Flag was subjected to different conditions before staining with Flag and γH2AX antibodies. Nuclei were stained with DAPI. The NCI-H1563 were not transduced to express A1 and exposed to dox (Mock), transduced to express A1 and exposed to dox and uracil DNA glycosylase inhibitor (Induced+UGI), transduced to express A1 and not exposed to dox (Uninduced), and transduced to express A1 and exposed to dox (Induced). These conditions were carried out after ( A and B ) 24 h and ( C and D ) 48 h. (B andD) Histogram shows the numbers of foci/cell in bins of 1–5 (5), 6–10 (10), 11–15 (15) and 16 or more (More). The experiment was independently repeated in triplicate. Representative images are shown.

    Techniques Used: Expressing, Staining

    6) Product Images from "A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe"

    Article Title: A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh851

    DNA glycosylase activities in cell extracts from S.pombe . ( A ) Cleavage of A/8oxoG-containing duplex DNA. An aliquot of 0.5 and 2.5 μg of protein extract from wild-type or nth1 cells, 20 ng MutY from E.coli and 10 ng purified Apn1 from S.cerevisiae , or 10 ng Apn1 only, were incubated with 10 fmol of a 24mer 32 P-labelled oligodeoxyribonucleotide harbouring an A opposite 8oxoG in the presence of 5 mM Mg 2+ . Strand cleavage was analysed by 20% denaturing PAGE and phosphorimaging. ( B ) MutY activity in wild-type and nth1 extracts. Protein extracts (2.5 µg) from wild-type or nth1 cells were incubated with A:8oxoG DNA (as in A) and subsequently treated with 100 mM NaOH. Relative cleavage was quantified with the ImageQuaNT software. ( C ) Uracil removing and nicking activity in wild-type protein extracts of S.pombe . Whole cell extract (0.5 μg) was incubated with 10 fmol of a 24 bp oligodeoxyribonucleotide containing a single uracil residue (opposite A) at position 14 with or without S.pombe Nth1 (2, 5 or 10 ng) or S.cerevisiae Apn1 (100, 250 or 500 pg) for 30 min at 37°C. Similar experiments with Udg (NEB) were also included as indicated. The reaction products were separated on a polyacrylamide gel and bands were detected by phosphorimaging.
    Figure Legend Snippet: DNA glycosylase activities in cell extracts from S.pombe . ( A ) Cleavage of A/8oxoG-containing duplex DNA. An aliquot of 0.5 and 2.5 μg of protein extract from wild-type or nth1 cells, 20 ng MutY from E.coli and 10 ng purified Apn1 from S.cerevisiae , or 10 ng Apn1 only, were incubated with 10 fmol of a 24mer 32 P-labelled oligodeoxyribonucleotide harbouring an A opposite 8oxoG in the presence of 5 mM Mg 2+ . Strand cleavage was analysed by 20% denaturing PAGE and phosphorimaging. ( B ) MutY activity in wild-type and nth1 extracts. Protein extracts (2.5 µg) from wild-type or nth1 cells were incubated with A:8oxoG DNA (as in A) and subsequently treated with 100 mM NaOH. Relative cleavage was quantified with the ImageQuaNT software. ( C ) Uracil removing and nicking activity in wild-type protein extracts of S.pombe . Whole cell extract (0.5 μg) was incubated with 10 fmol of a 24 bp oligodeoxyribonucleotide containing a single uracil residue (opposite A) at position 14 with or without S.pombe Nth1 (2, 5 or 10 ng) or S.cerevisiae Apn1 (100, 250 or 500 pg) for 30 min at 37°C. Similar experiments with Udg (NEB) were also included as indicated. The reaction products were separated on a polyacrylamide gel and bands were detected by phosphorimaging.

    Techniques Used: Purification, Incubation, Polyacrylamide Gel Electrophoresis, Activity Assay, Software

    7) Product Images from "Reduced Shmt2 expression impairs mitochondrial folate accumulation and respiration, and leads to uracil accumulation in mouse mitochondrial DNA"

    Article Title: Reduced Shmt2 expression impairs mitochondrial folate accumulation and respiration, and leads to uracil accumulation in mouse mitochondrial DNA

    Journal: bioRxiv

    doi: 10.1101/2021.04.12.439270

    Uracil content in Shmt2 +/+ and Shmt2 +/- mouse liver mtDNA. A) Uracil content in Shmt2 +/+ and Shmt2 +/- mouse liver mtDNA from male mice consuming the C or FD diet for 7 weeks and B) Uracil content both before and after UDG treatment. The content represents total uracil in each region. Two-way ANOVA with Tukey’s post-hoc analysis was used to determine diet by genotype interaction and main effects of diet and genotype and data are normalized to the Shmt2 +/+ mice on control diet in panel A. Statistical significant was determined for genotype (*), diet (**), and diet by genotype interaction (***). Levels not connected by the same letter are significantly different, n = 3 per group. Student’s t-tests were used to analyze data in panel B. Data represent means ± SD. P values ≤ 0.05 were considered significantly different. n = 2 per group. * indicates statistical significant. C, control; FD, folate deficient; SHMT2, serine hydroxymethyltransferase 2; UDG, uracil dna glycosylase.
    Figure Legend Snippet: Uracil content in Shmt2 +/+ and Shmt2 +/- mouse liver mtDNA. A) Uracil content in Shmt2 +/+ and Shmt2 +/- mouse liver mtDNA from male mice consuming the C or FD diet for 7 weeks and B) Uracil content both before and after UDG treatment. The content represents total uracil in each region. Two-way ANOVA with Tukey’s post-hoc analysis was used to determine diet by genotype interaction and main effects of diet and genotype and data are normalized to the Shmt2 +/+ mice on control diet in panel A. Statistical significant was determined for genotype (*), diet (**), and diet by genotype interaction (***). Levels not connected by the same letter are significantly different, n = 3 per group. Student’s t-tests were used to analyze data in panel B. Data represent means ± SD. P values ≤ 0.05 were considered significantly different. n = 2 per group. * indicates statistical significant. C, control; FD, folate deficient; SHMT2, serine hydroxymethyltransferase 2; UDG, uracil dna glycosylase.

    Techniques Used: Mouse Assay

    8) Product Images from "Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroiminohydantoin and guanidinohydantoin"

    Article Title: Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroiminohydantoin and guanidinohydantoin

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2013.04.026

    3.4 Human NEIL3 acts mainly as a monofunctional DNA glycosylase with highest affinity for the hydantoin lesions Sp and Gh
    Figure Legend Snippet: 3.4 Human NEIL3 acts mainly as a monofunctional DNA glycosylase with highest affinity for the hydantoin lesions Sp and Gh

    Techniques Used:

    Residues involved in DNA glycosylase and AP lyase activity of human NEIL3
    Figure Legend Snippet: Residues involved in DNA glycosylase and AP lyase activity of human NEIL3

    Techniques Used: Activity Assay

    9) Product Images from "Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine"

    Article Title: Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2012.06.004

    Glycosylase/lyase activity assays and activity profile on γ-irradiated DNA of wild-type EcoFpg and EcoFpgΔ213-229. (A) The glycosylase assay was performed by incubating 10 nM of double-stranded substrate containing 8-oxoG:C, MeFapy:C,
    Figure Legend Snippet: Glycosylase/lyase activity assays and activity profile on γ-irradiated DNA of wild-type EcoFpg and EcoFpgΔ213-229. (A) The glycosylase assay was performed by incubating 10 nM of double-stranded substrate containing 8-oxoG:C, MeFapy:C,

    Techniques Used: Activity Assay, Irradiation

    10) Product Images from "T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

    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.
    Figure Legend 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.

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

    11) Product Images from "Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis"

    Article Title: Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2019.102767

    TkoAGOG is a bifunctional 8oxoG DNA glycosylase. A , Proposed enzymatic mechanism of a bifunctional 8oxoG DNA glycosylase, where 8oxoG nucleobase loss is followed by a Schiff base intermediate that undergoes β-elimination and for some DNA glycosylases such as Fpg further δ-elimination. The confirmed TkoAGOG mechanism is boxed in grey B , A 60-nt, 5’-FAM (blue), 3’-ROX (red) labeled dsDNA substrate with a centralized 8oxoG:C (or dU:G) was incubated for 30 min with either TkoAGOG at 65 °C, Fpg at 37 °C, or UDG/EndoIII at 37 °C and quenched with Formamide + EDTA, allowing for visualization of the base excision glycosylase and AP lyase activities. C , The conversion of 60-nt substrate to the 5’-FAM and 3’-ROX products after incubation with TkoAGOG, Fpg, or UDG/EndoIII.
    Figure Legend Snippet: TkoAGOG is a bifunctional 8oxoG DNA glycosylase. A , Proposed enzymatic mechanism of a bifunctional 8oxoG DNA glycosylase, where 8oxoG nucleobase loss is followed by a Schiff base intermediate that undergoes β-elimination and for some DNA glycosylases such as Fpg further δ-elimination. The confirmed TkoAGOG mechanism is boxed in grey B , A 60-nt, 5’-FAM (blue), 3’-ROX (red) labeled dsDNA substrate with a centralized 8oxoG:C (or dU:G) was incubated for 30 min with either TkoAGOG at 65 °C, Fpg at 37 °C, or UDG/EndoIII at 37 °C and quenched with Formamide + EDTA, allowing for visualization of the base excision glycosylase and AP lyase activities. C , The conversion of 60-nt substrate to the 5’-FAM and 3’-ROX products after incubation with TkoAGOG, Fpg, or UDG/EndoIII.

    Techniques Used: Labeling, Incubation

    Models of 8oxoG long-patch BER in each Domain of life. 8oxo BER is initiated by a DNA glycosylase (Ogg1, AGOG or Fpg). In Eukarya and Archaea, downstream repair is carried out by an AP endonuclease (APE1 or EndoIV), DNA polymerase, Fen1 and DNA ligase. In Bacteria, downstream repair requires ExoIII or EndoIV, Pol I, and DNA ligase.
    Figure Legend Snippet: Models of 8oxoG long-patch BER in each Domain of life. 8oxo BER is initiated by a DNA glycosylase (Ogg1, AGOG or Fpg). In Eukarya and Archaea, downstream repair is carried out by an AP endonuclease (APE1 or EndoIV), DNA polymerase, Fen1 and DNA ligase. In Bacteria, downstream repair requires ExoIII or EndoIV, Pol I, and DNA ligase.

    Techniques Used:

    12) Product Images from "T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

    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.
    Figure Legend 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.

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

    13) Product Images from "T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

    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.
    Figure Legend 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.

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

    14) Product Images from "Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells"

    Article Title: Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.333963

    Involvement of Tdp1 for repair of abasic (AP) sites. A , scheme for the conversion of MMS- and H 2 O 2 -induced DNA damage into the substrates for Tdp1. DNA adducts like methylated base and 8-oxoguanine ( 8-oxo ) are converted into AP sites by DNA glycosylase.
    Figure Legend Snippet: Involvement of Tdp1 for repair of abasic (AP) sites. A , scheme for the conversion of MMS- and H 2 O 2 -induced DNA damage into the substrates for Tdp1. DNA adducts like methylated base and 8-oxoguanine ( 8-oxo ) are converted into AP sites by DNA glycosylase.

    Techniques Used: Methylation

    15) Product Images from "Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage"

    Article Title: Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn118

    True color fluorescence oligonucleotide assay. ( I ) Scheme for construction of dual-color fluorescently labeled oligonucleotides. The 51mer A strand contains a single uracil, whereas the opposing strand is synthesized from a central cassette (Bb, 21 bp) containing one of a number of lesion configurations, and two flanking sequences, Ba and Bc, each 15 bp. In the example shown, A contains one uracil residue, and is labeled at its 5′ end with 6-FAM; Ba is 3′ end-labeled with TAMRA, and the central Bb cassette contains one uracil residue. The components are annealed, ligated and treated with uracil DNA glycosylase to convert the uracil moieties to abasic sites. The action of Ape1 on the construct is then assessed. ( II ) True color denaturing gel (adjacent segments of the same gel, separated for clarity) with fluorescence of intact and Ape1-cleaved oligonucleotides. Constructs and pairs of gel lanes showing substrates (Lanes 1, 3 and 5) and products (Lanes 2, 4 and 6). Lanes 1 and 2: 51mer A1•B−5, where A1 is 5′-labeled with 6-FAM, and B-5 is 3″ TAMRA-labeled. Lane 1 intact substrate plus free, unligated TAMRA-labeled Ba); Lane 2, products of Ape1 cleavage of A1•B−5: 3′ end of B- TAMRA, 5′ end of A-FAM) plus unligated Ba. Lanes 3 and 4: A1•B−5 containing unlabelled A1 and dually labeled B-5 (3′ TAMRA and 5′ 6-FAM). Lane 3, intact substrate, a small quantity of the partial ligation product BaBb, plus unligated TAMRA-labeled Ba and 6-FAM-labeled Bc. Lane 4, Ape cleavage products: 3′ end of B, 5′ end of B plus Ba and Bc as in Lane 3. Lanes 5 and 6, Substrate and products as in Lanes 3 and 4, but Bc was 5′-labeled with JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, light green) and 3′- labeled with TAMRA.
    Figure Legend Snippet: True color fluorescence oligonucleotide assay. ( I ) Scheme for construction of dual-color fluorescently labeled oligonucleotides. The 51mer A strand contains a single uracil, whereas the opposing strand is synthesized from a central cassette (Bb, 21 bp) containing one of a number of lesion configurations, and two flanking sequences, Ba and Bc, each 15 bp. In the example shown, A contains one uracil residue, and is labeled at its 5′ end with 6-FAM; Ba is 3′ end-labeled with TAMRA, and the central Bb cassette contains one uracil residue. The components are annealed, ligated and treated with uracil DNA glycosylase to convert the uracil moieties to abasic sites. The action of Ape1 on the construct is then assessed. ( II ) True color denaturing gel (adjacent segments of the same gel, separated for clarity) with fluorescence of intact and Ape1-cleaved oligonucleotides. Constructs and pairs of gel lanes showing substrates (Lanes 1, 3 and 5) and products (Lanes 2, 4 and 6). Lanes 1 and 2: 51mer A1•B−5, where A1 is 5′-labeled with 6-FAM, and B-5 is 3″ TAMRA-labeled. Lane 1 intact substrate plus free, unligated TAMRA-labeled Ba); Lane 2, products of Ape1 cleavage of A1•B−5: 3′ end of B- TAMRA, 5′ end of A-FAM) plus unligated Ba. Lanes 3 and 4: A1•B−5 containing unlabelled A1 and dually labeled B-5 (3′ TAMRA and 5′ 6-FAM). Lane 3, intact substrate, a small quantity of the partial ligation product BaBb, plus unligated TAMRA-labeled Ba and 6-FAM-labeled Bc. Lane 4, Ape cleavage products: 3′ end of B, 5′ end of B plus Ba and Bc as in Lane 3. Lanes 5 and 6, Substrate and products as in Lanes 3 and 4, but Bc was 5′-labeled with JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, light green) and 3′- labeled with TAMRA.

    Techniques Used: Fluorescence, Oligonucleotide Assay, Labeling, Synthesized, Construct, Ligation

    16) Product Images from "Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism"

    Article Title: Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006201

    Cleavage reactions with transposase mutants and an abasic substrate. Transpososomes were first assembled in the absence of divalent metal ions. The cleavage reaction was initiated by the addition of MgCl 2 at time zero. Aliquots were withdrawn at the indicated times and the reaction halted by the addition of EDTA and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography on a phosphoimager. The DNA substrates were labeled at both 5′-ends so that all three phosphoryl transfer reactions could be observed in a single experiment. The steps of the cleavage reaction are shown in panel A of the figure below the gel panel. The flanking DNA is to the left and the transposon arm to the right of the half bracket that indicates the location of the transposon end. The positions of the radioactive labels are indicated by the asterisks. Since the reactions are analyzed on denaturing gels, the unlabeled DNA strands, illustrated in grey, are not detected in the autoradiograms. The identity of each band is indicated to the right of the gel in panel A. Bands I and IV each represent a single product of the reaction as indicated. Bands II and III each represent mixtures of more than one co-migrating product and/or substrate as indicated. A B Cleavage reactions of wild type and abasic DNA substrates. The diagonal slashes indicate regions of the gels that have been removed because they contain no relevant information. Unaltered images of the gels are provided in Figure S1 . The identity of the products are indicated next to each band: Band I is the hairpin intermediate; Band II consists the unreacted substrate plus the top strand of the nicked product; Band III contains the bottom strand of the nicked product and the bottom strand of the cleaved transposon end (the resolved hairpin); Band IV contains the top strand of the cleaved flanking DNA that is released upon hairpin formation. In panel B the substrate has an abasic residue at position +2 of the top strand. This was prepared by incorporating a uracil residue at that position by PCR and subsequently treating the substrate with uracil glycosylase. This approach was preferred over one in which the abasic site could have been incorporated during oligonucleotide synthesis. Tn 10 transposon arms are folded during assembly of the transpososome [32] , [39] , [40] , and the DNA fragments required are too long for convenient oligonucleotide synthesis. C-F Quantification of cleavage intermediates. The respective products are plotted as a percentage of the total substrate in the reaction. The amount of each intermediate present at each time point is indicated by the shading within the column. None of the conditions tested severely inhibit the nicking step of the reaction. Sixty minutes is sufficient time for all of the transpososome complexes present at the start of the reaction to achieve the first nick. The height of the column at the 60 minute time point is therefore equivalent to the efficiency of transposome assembly, which varied over a 3-fold range in the reactions presented in this experiment. Bands I and IV (corresponding to the hairpin and cleaved top strand, respectively) are unique and unambiguous products of the reaction and can be quantified directly from the gel by phosphorimager analysis. Other intermediates and/or substrate comigrate and therefore can not be quantified directly. They were calculated as follows: first strand cleavage (first nick) = Band III - (Band IV - Band I). Hairpin resolution = Band IV - Band I. These calculations rely on equal labeling efficiency at either end of the substrate. To determine the efficiency of labeling an aliquot of the substrate was cleaved into two parts by NdeI, and the ratio of label incorporated at each end of the fragment was determined by phosphoimager analysis. This ratio was used to adjust all quantifications described above.
    Figure Legend Snippet: Cleavage reactions with transposase mutants and an abasic substrate. Transpososomes were first assembled in the absence of divalent metal ions. The cleavage reaction was initiated by the addition of MgCl 2 at time zero. Aliquots were withdrawn at the indicated times and the reaction halted by the addition of EDTA and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography on a phosphoimager. The DNA substrates were labeled at both 5′-ends so that all three phosphoryl transfer reactions could be observed in a single experiment. The steps of the cleavage reaction are shown in panel A of the figure below the gel panel. The flanking DNA is to the left and the transposon arm to the right of the half bracket that indicates the location of the transposon end. The positions of the radioactive labels are indicated by the asterisks. Since the reactions are analyzed on denaturing gels, the unlabeled DNA strands, illustrated in grey, are not detected in the autoradiograms. The identity of each band is indicated to the right of the gel in panel A. Bands I and IV each represent a single product of the reaction as indicated. Bands II and III each represent mixtures of more than one co-migrating product and/or substrate as indicated. A B Cleavage reactions of wild type and abasic DNA substrates. The diagonal slashes indicate regions of the gels that have been removed because they contain no relevant information. Unaltered images of the gels are provided in Figure S1 . The identity of the products are indicated next to each band: Band I is the hairpin intermediate; Band II consists the unreacted substrate plus the top strand of the nicked product; Band III contains the bottom strand of the nicked product and the bottom strand of the cleaved transposon end (the resolved hairpin); Band IV contains the top strand of the cleaved flanking DNA that is released upon hairpin formation. In panel B the substrate has an abasic residue at position +2 of the top strand. This was prepared by incorporating a uracil residue at that position by PCR and subsequently treating the substrate with uracil glycosylase. This approach was preferred over one in which the abasic site could have been incorporated during oligonucleotide synthesis. Tn 10 transposon arms are folded during assembly of the transpososome [32] , [39] , [40] , and the DNA fragments required are too long for convenient oligonucleotide synthesis. C-F Quantification of cleavage intermediates. The respective products are plotted as a percentage of the total substrate in the reaction. The amount of each intermediate present at each time point is indicated by the shading within the column. None of the conditions tested severely inhibit the nicking step of the reaction. Sixty minutes is sufficient time for all of the transpososome complexes present at the start of the reaction to achieve the first nick. The height of the column at the 60 minute time point is therefore equivalent to the efficiency of transposome assembly, which varied over a 3-fold range in the reactions presented in this experiment. Bands I and IV (corresponding to the hairpin and cleaved top strand, respectively) are unique and unambiguous products of the reaction and can be quantified directly from the gel by phosphorimager analysis. Other intermediates and/or substrate comigrate and therefore can not be quantified directly. They were calculated as follows: first strand cleavage (first nick) = Band III - (Band IV - Band I). Hairpin resolution = Band IV - Band I. These calculations rely on equal labeling efficiency at either end of the substrate. To determine the efficiency of labeling an aliquot of the substrate was cleaved into two parts by NdeI, and the ratio of label incorporated at each end of the fragment was determined by phosphoimager analysis. This ratio was used to adjust all quantifications described above.

    Techniques Used: DNA Sequencing, Autoradiography, Labeling, Polymerase Chain Reaction, Oligonucleotide Synthesis

    17) Product Images from "Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage"

    Article Title: Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn118

    True color fluorescence oligonucleotide assay. ( I ) Scheme for construction of dual-color fluorescently labeled oligonucleotides. The 51mer A strand contains a single uracil, whereas the opposing strand is synthesized from a central cassette (Bb, 21 bp) containing one of a number of lesion configurations, and two flanking sequences, Ba and Bc, each 15 bp. In the example shown, A contains one uracil residue, and is labeled at its 5′ end with 6-FAM; Ba is 3′ end-labeled with TAMRA, and the central Bb cassette contains one uracil residue. The components are annealed, ligated and treated with uracil DNA glycosylase to convert the uracil moieties to abasic sites. The action of Ape1 on the construct is then assessed. ( II ) True color denaturing gel (adjacent segments of the same gel, separated for clarity) with fluorescence of intact and Ape1-cleaved oligonucleotides. Constructs and pairs of gel lanes showing substrates (Lanes 1, 3 and 5) and products (Lanes 2, 4 and 6). Lanes 1 and 2: 51mer A1•B−5, where A1 is 5′-labeled with 6-FAM, and B-5 is 3″ TAMRA-labeled. Lane 1 intact substrate plus free, unligated TAMRA-labeled Ba); Lane 2, products of Ape1 cleavage of A1•B−5: 3′ end of B- TAMRA, 5′ end of A-FAM) plus unligated Ba. Lanes 3 and 4: A1•B−5 containing unlabelled A1 and dually labeled B-5 (3′ TAMRA and 5′ 6-FAM). Lane 3, intact substrate, a small quantity of the partial ligation product BaBb, plus unligated TAMRA-labeled Ba and 6-FAM-labeled Bc. Lane 4, Ape cleavage products: 3′ end of B, 5′ end of B plus Ba and Bc as in Lane 3. Lanes 5 and 6, Substrate and products as in Lanes 3 and 4, but Bc was 5′-labeled with JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, light green) and 3′- labeled with TAMRA.
    Figure Legend Snippet: True color fluorescence oligonucleotide assay. ( I ) Scheme for construction of dual-color fluorescently labeled oligonucleotides. The 51mer A strand contains a single uracil, whereas the opposing strand is synthesized from a central cassette (Bb, 21 bp) containing one of a number of lesion configurations, and two flanking sequences, Ba and Bc, each 15 bp. In the example shown, A contains one uracil residue, and is labeled at its 5′ end with 6-FAM; Ba is 3′ end-labeled with TAMRA, and the central Bb cassette contains one uracil residue. The components are annealed, ligated and treated with uracil DNA glycosylase to convert the uracil moieties to abasic sites. The action of Ape1 on the construct is then assessed. ( II ) True color denaturing gel (adjacent segments of the same gel, separated for clarity) with fluorescence of intact and Ape1-cleaved oligonucleotides. Constructs and pairs of gel lanes showing substrates (Lanes 1, 3 and 5) and products (Lanes 2, 4 and 6). Lanes 1 and 2: 51mer A1•B−5, where A1 is 5′-labeled with 6-FAM, and B-5 is 3″ TAMRA-labeled. Lane 1 intact substrate plus free, unligated TAMRA-labeled Ba); Lane 2, products of Ape1 cleavage of A1•B−5: 3′ end of B- TAMRA, 5′ end of A-FAM) plus unligated Ba. Lanes 3 and 4: A1•B−5 containing unlabelled A1 and dually labeled B-5 (3′ TAMRA and 5′ 6-FAM). Lane 3, intact substrate, a small quantity of the partial ligation product BaBb, plus unligated TAMRA-labeled Ba and 6-FAM-labeled Bc. Lane 4, Ape cleavage products: 3′ end of B, 5′ end of B plus Ba and Bc as in Lane 3. Lanes 5 and 6, Substrate and products as in Lanes 3 and 4, but Bc was 5′-labeled with JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, light green) and 3′- labeled with TAMRA.

    Techniques Used: Fluorescence, Oligonucleotide Assay, Labeling, Synthesized, Construct, Ligation

    18) Product Images from "DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY"

    Article Title: DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2017.01.002

    Cisplatin cytotoxicity and effect on glycosylase activity (A) Colony survival assay in MDA-MB-231 cells following UNG and SMUG1 knockdown: shControl (open circles), shUNG (closed triangles), shSMUG1 (closed circles) and shUNG + shSMUG1 (open squares). Results are represented as mean ± SE from 3 independent experiments. Cells were transfected with shRNA directed against UNG and SMUG1. (B) Colony survival assay in MDA-MB-231 cells following MBD4 knockdown with shControl (open circles) and shMBD4 (closed triangles). shRNA transfected cells were treated with increasing doses of cisplatin and cytotoxicity. Results are represented as mean ± SE from 3 independent experiments. (C) In vitro glycosylase assay, DNA (5nM) was incubated with either pure enzyme or HeLa extract. Lane 1, undamaged DNA alone.; lane 2, undamaged DNA treated with UDG and APE1 to generate a 19 mer product; lane 3, undamaged DNA substrate treated with UDG, APE1 and 1 unit of UGI; lane 4, undamaged DNA incubated with HeLa extract; lane 5 reactions in which HeLa extract was preincubated with 1 unit of UGI before adding the undamaged DNA substrate. Lanes 6–10 follow the same set up as lanes 1–5, but with ICL DNA substrate. Both undamaged and ICL substrates contain a central uracil and a 3′ Cy3 label. M is a 21-nt marker.
    Figure Legend Snippet: Cisplatin cytotoxicity and effect on glycosylase activity (A) Colony survival assay in MDA-MB-231 cells following UNG and SMUG1 knockdown: shControl (open circles), shUNG (closed triangles), shSMUG1 (closed circles) and shUNG + shSMUG1 (open squares). Results are represented as mean ± SE from 3 independent experiments. Cells were transfected with shRNA directed against UNG and SMUG1. (B) Colony survival assay in MDA-MB-231 cells following MBD4 knockdown with shControl (open circles) and shMBD4 (closed triangles). shRNA transfected cells were treated with increasing doses of cisplatin and cytotoxicity. Results are represented as mean ± SE from 3 independent experiments. (C) In vitro glycosylase assay, DNA (5nM) was incubated with either pure enzyme or HeLa extract. Lane 1, undamaged DNA alone.; lane 2, undamaged DNA treated with UDG and APE1 to generate a 19 mer product; lane 3, undamaged DNA substrate treated with UDG, APE1 and 1 unit of UGI; lane 4, undamaged DNA incubated with HeLa extract; lane 5 reactions in which HeLa extract was preincubated with 1 unit of UGI before adding the undamaged DNA substrate. Lanes 6–10 follow the same set up as lanes 1–5, but with ICL DNA substrate. Both undamaged and ICL substrates contain a central uracil and a 3′ Cy3 label. M is a 21-nt marker.

    Techniques Used: Activity Assay, Clonogenic Cell Survival Assay, Multiple Displacement Amplification, Transfection, shRNA, In Vitro, Incubation, Marker

    19) Product Images from "Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma"

    Article Title: Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgp118

    Analysis of hOGG1 variants. ( A ) 8oxoG DNA glycosylase activity of S31P compared with WT hOGG1. A total of 3 and 10 ng enzymes were incubated with an 8oxoG:C oligonucleotide at 37°C for 30 min before cleavage of the phosphodiester backbone by NaOH. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand and C = cleavage product). ( B ) DNA binding properties of hOGG1 WT and S31P. WT and S31P hOGG1 (10, 30 and 100 ng) were incubated with 8oxoG:C DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.
    Figure Legend Snippet: Analysis of hOGG1 variants. ( A ) 8oxoG DNA glycosylase activity of S31P compared with WT hOGG1. A total of 3 and 10 ng enzymes were incubated with an 8oxoG:C oligonucleotide at 37°C for 30 min before cleavage of the phosphodiester backbone by NaOH. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand and C = cleavage product). ( B ) DNA binding properties of hOGG1 WT and S31P. WT and S31P hOGG1 (10, 30 and 100 ng) were incubated with 8oxoG:C DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.

    Techniques Used: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay

    Analysis of hMYH variants. ( A ) Adenine DNA glycosylase activities of hMYH WT, R260Q, H434D and S501F variants were measured by incubating the respective proteins (18 ng) with a duplex oligodeoxyribonucleotide containing a single A:8oxoG or A:G basepair at 37°C for 30 min. Strand cleavage after NaOH treatment was analyzed by 20% polyacrylamide gel electrophoresis and phosphorimaging (I = intact strand and C = cleavage product). ( B ) Different amounts (0.6–240 ng) of hMYH WT (□), R260Q (▴), H434D (X) and S501F (*) were assayed for A:8oxoG DNA glycosylase activities and percentage strand cleavage quantified with ImageQuant. Extract from Escherichia coli cells expressing empty vector and purified similarly as hMYH was used to measure the background level (⧫). ( C ) DNA binding properties of hMYH WT, R260Q, H434D and S501F (24 ng) to substrates containing A:8oxoG (left panel) or A:G (right panel). After incubation on ice, DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.
    Figure Legend Snippet: Analysis of hMYH variants. ( A ) Adenine DNA glycosylase activities of hMYH WT, R260Q, H434D and S501F variants were measured by incubating the respective proteins (18 ng) with a duplex oligodeoxyribonucleotide containing a single A:8oxoG or A:G basepair at 37°C for 30 min. Strand cleavage after NaOH treatment was analyzed by 20% polyacrylamide gel electrophoresis and phosphorimaging (I = intact strand and C = cleavage product). ( B ) Different amounts (0.6–240 ng) of hMYH WT (□), R260Q (▴), H434D (X) and S501F (*) were assayed for A:8oxoG DNA glycosylase activities and percentage strand cleavage quantified with ImageQuant. Extract from Escherichia coli cells expressing empty vector and purified similarly as hMYH was used to measure the background level (⧫). ( C ) DNA binding properties of hMYH WT, R260Q, H434D and S501F (24 ng) to substrates containing A:8oxoG (left panel) or A:G (right panel). After incubation on ice, DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Expressing, Plasmid Preparation, Purification, Binding Assay, Incubation

    Analysis of NEIL1 variants. ( A ) DNA glycosylase activity of G83D compared with WT NEIL1. Enzymes (2, 5, 10 and 20 ng) were incubated with different oligonucleotide substrates as indicated at 37°C for 30 min. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand, C = cleavage product, β = β elimination, δ = δ elimination cleavage, ss = single strand). ( B ) FaPy DNA glycosylase activity of NEIL1 WT (⧫) and G83D (▪). Enzymes (3, 10, 30 and 100 ng) were assayed for removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC). ( C ) DNA binding properties of NEIL1 WT and G83D. NEIL1 WT and G83D (20, 50 and 100 ng) were incubated with 5ohC:G DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein. ( D ) Nuclear localization of NEIL1 G83D and E181K. Asynchronous growing HeLa S3 cells were transiently transfected with constructs expressing NEIL1-EGFP, NEIL1G83D-EGFP or NEIL1E181K-EGFP. Cells were imaged directly by fluorescence microscopy for EGFP detection. DNA was stained with Hoechst 33342.
    Figure Legend Snippet: Analysis of NEIL1 variants. ( A ) DNA glycosylase activity of G83D compared with WT NEIL1. Enzymes (2, 5, 10 and 20 ng) were incubated with different oligonucleotide substrates as indicated at 37°C for 30 min. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand, C = cleavage product, β = β elimination, δ = δ elimination cleavage, ss = single strand). ( B ) FaPy DNA glycosylase activity of NEIL1 WT (⧫) and G83D (▪). Enzymes (3, 10, 30 and 100 ng) were assayed for removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC). ( C ) DNA binding properties of NEIL1 WT and G83D. NEIL1 WT and G83D (20, 50 and 100 ng) were incubated with 5ohC:G DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein. ( D ) Nuclear localization of NEIL1 G83D and E181K. Asynchronous growing HeLa S3 cells were transiently transfected with constructs expressing NEIL1-EGFP, NEIL1G83D-EGFP or NEIL1E181K-EGFP. Cells were imaged directly by fluorescence microscopy for EGFP detection. DNA was stained with Hoechst 33342.

    Techniques Used: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay, Transfection, Construct, Expressing, Fluorescence, Microscopy, Staining

    20) Product Images from "The ubiquitin ligase RFWD3 is required for translesion DNA synthesis"

    Article Title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2020.11.029

    RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see Discussion ).
    Figure Legend Snippet: RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see Discussion ).

    Techniques Used: Incubation, DNA Synthesis

    21) Product Images from "Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases"

    Article Title: Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2008.12.013

    DNA glycosylase/lyase activities of AthFpg and CalFpg. Double-stranded substrates (25 nM) were incubated with the appropriate control enzymes (25 nM) (EcoFpg and hOGG1 for 8oxoG:C, EcoNth, EcoNei and hNEIL1 for double-stranded pyrimidines, EcoFpg, EcoNei and hNEIL1 for Gh:C, Sp1:C and Sp2:C); 2.5, 25 or 250 nM AtFpg and CalFpg as described in Section 2.
    Figure Legend Snippet: DNA glycosylase/lyase activities of AthFpg and CalFpg. Double-stranded substrates (25 nM) were incubated with the appropriate control enzymes (25 nM) (EcoFpg and hOGG1 for 8oxoG:C, EcoNth, EcoNei and hNEIL1 for double-stranded pyrimidines, EcoFpg, EcoNei and hNEIL1 for Gh:C, Sp1:C and Sp2:C); 2.5, 25 or 250 nM AtFpg and CalFpg as described in Section 2.

    Techniques Used: Incubation

    22) Product Images from "Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases"

    Article Title: Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2013.09.004

    Role of the void-filling Met72 and adjacent His73 in lesion excision. Glycosylase assays with double-stranded Sp1:C (A) and ssSp1 (B) where the DNA substrate (20 nM) was combined with 16 nM of either WT or mutant MvNei2. WT MvNei2 is displayed as circles.
    Figure Legend Snippet: Role of the void-filling Met72 and adjacent His73 in lesion excision. Glycosylase assays with double-stranded Sp1:C (A) and ssSp1 (B) where the DNA substrate (20 nM) was combined with 16 nM of either WT or mutant MvNei2. WT MvNei2 is displayed as circles.

    Techniques Used: Mutagenesis

    23) Product Images from "APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A"

    Article Title: APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1201

    Characterization of A1 RNA binding and ssDNA deamination activity. ( A ) Determination of nucleic acids that co-purify with A1. Experimental outline (sketch) shown alongside visualization of samples after denaturing gel electrophoresis. ( B ) Determination of the preferential deamination motif of A1 using the Uracil DNA Glycosylase (UDG) assay (sketch, left). Deamination of an 85 nt ssDNA with deaminated cytosines spaced 30 nt apart and an internal fluorescein label (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of 67 and 48 nt fragments, respectively; double deamination of both residues results in a 30 nt fragment. The 85 nt ssDNA substrates contained different deamination motifs, as labeled above the gel, to determine the optimal substrate. The specific activity (S.A.) was measured as pmol substrate deaminated/μg enzyme/min (pmol/μg/min) ( C ) Deamination of a 118 nt ssDNA substrate with deamination motifs spaced 63 nt apart and an internal fluorescein level (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of labeled 110 and 81 nt fragments, respectively; double deamination of both C residues on the same ssDNA results in a 63 nt fragment. A1 was either treated or not treated with RNase A. Analysis to determine a processivity factor (P.F.) is illustrated in the sketch, left. (B and C) The measurements of S.A., P.F., or standard deviation (S.D.) from three independent experiments are indicated below the gel.
    Figure Legend Snippet: Characterization of A1 RNA binding and ssDNA deamination activity. ( A ) Determination of nucleic acids that co-purify with A1. Experimental outline (sketch) shown alongside visualization of samples after denaturing gel electrophoresis. ( B ) Determination of the preferential deamination motif of A1 using the Uracil DNA Glycosylase (UDG) assay (sketch, left). Deamination of an 85 nt ssDNA with deaminated cytosines spaced 30 nt apart and an internal fluorescein label (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of 67 and 48 nt fragments, respectively; double deamination of both residues results in a 30 nt fragment. The 85 nt ssDNA substrates contained different deamination motifs, as labeled above the gel, to determine the optimal substrate. The specific activity (S.A.) was measured as pmol substrate deaminated/μg enzyme/min (pmol/μg/min) ( C ) Deamination of a 118 nt ssDNA substrate with deamination motifs spaced 63 nt apart and an internal fluorescein level (sketch, above gel). Single deaminations of the 5′C and 3′C are detected as the appearance of labeled 110 and 81 nt fragments, respectively; double deamination of both C residues on the same ssDNA results in a 63 nt fragment. A1 was either treated or not treated with RNase A. Analysis to determine a processivity factor (P.F.) is illustrated in the sketch, left. (B and C) The measurements of S.A., P.F., or standard deviation (S.D.) from three independent experiments are indicated below the gel.

    Techniques Used: RNA Binding Assay, Activity Assay, Nucleic Acid Electrophoresis, Labeling, Standard Deviation

    Effect of A1 expression on γH2AX foci. Cancerous lung cell line NCI-H1563 with or without stable doxycycline (dox) inducible A1-Flag was subjected to different conditions before staining with Flag and γH2AX antibodies. Nuclei were stained with DAPI. The NCI-H1563 were not transduced to express A1 and exposed to dox (Mock), transduced to express A1 and exposed to dox and uracil DNA glycosylase inhibitor (Induced+UGI), transduced to express A1 and not exposed to dox (Uninduced), and transduced to express A1 and exposed to dox (Induced). These conditions were carried out after ( A and B ) 24 h and ( C and D ) 48 h. (B andD) Histogram shows the numbers of foci/cell in bins of 1–5 (5), 6–10 (10), 11–15 (15) and 16 or more (More). The experiment was independently repeated in triplicate. Representative images are shown.
    Figure Legend Snippet: Effect of A1 expression on γH2AX foci. Cancerous lung cell line NCI-H1563 with or without stable doxycycline (dox) inducible A1-Flag was subjected to different conditions before staining with Flag and γH2AX antibodies. Nuclei were stained with DAPI. The NCI-H1563 were not transduced to express A1 and exposed to dox (Mock), transduced to express A1 and exposed to dox and uracil DNA glycosylase inhibitor (Induced+UGI), transduced to express A1 and not exposed to dox (Uninduced), and transduced to express A1 and exposed to dox (Induced). These conditions were carried out after ( A and B ) 24 h and ( C and D ) 48 h. (B andD) Histogram shows the numbers of foci/cell in bins of 1–5 (5), 6–10 (10), 11–15 (15) and 16 or more (More). The experiment was independently repeated in triplicate. Representative images are shown.

    Techniques Used: Expressing, Staining

    Related Articles

    other:

    Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity
    Article Snippet: uracil DNA glycosylase

    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: The PCR cycling and melting conditions were as follows; an initial incubation at 95°C for 15 mins, followed by 55 cycles of 95°C for 10 s, 60°C for 20 s, and 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 step. .. 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: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase
    Article Snippet: The PCR cycling and melting conditions were as follows; an initial incubation at 95°C for 15 mins, followed by 55 cycles of 95°C for 10 s, 60°C for 20 s, and 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 step. .. 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.

    Incubation:

    Article Title: Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA
    Article Snippet: Oligonucleotide deaminase activity assay Oligonucleotide deaminase activity assay was performed according to the published method . .. Briefly, 5′-Cy5-labeled oligonucleotide ( 5′-Cy5-TTTTTTTTTTTTTTTATCTTTTTTTTTTTACTTTTTTTTTTAAACCCAAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT ) was incubated with A3G in the presence of UDG (New England Biolabs). ..

    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.

    Purification:

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations
    Article Snippet: NEIL2 and APE1 glycosylase/lyase activity assay NEIL2 activity was measured as previously described ( ). .. Briefly, 0.5 pmol 32 P-labeled oligonucleotide substrate (35 nt or 51 nt) was mixed on ice with 1 μl UDG (1 U/μl) (NEB, M0280S), and purified NEIL2 (68 ng/µl) or APE1 (NEB, M0282S, 0.005 U/µl), and 1× NEBuffer 4 (50 mM KOAc, 20 mM Tris-Acetate, pH 7.9, 10 mM Mg(OAc)2 , 1 mM DTT) in a total reaction volume of 10 μl. .. Reactions were incubated at 37°C for 30 min. After PCI extraction and ethanol precipitation, samples were treated with equal volume of 2× Novex TBE-Urea Sample Buffer (Invitrogen).

    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.

    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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    New England Biolabs udg
    Schematic diagram showing the mechanism of the uracil DNA glycosylase-supplemented real-time reverse-transcription loop-mediated isothermal amplification <t>(UDG-rRT-LAMP)</t> assay. Blue LAMP amplicons represent <t>non-dUTP-incorporated</t> 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.
    Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Scientific Reports

    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

    doi: 10.1038/srep27605

    Figure Lengend 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.

    Article Snippet: The total reaction master mix volume was 18 μL consisting of 1.6 μM each of FIP and BIP primers, 0.2 μM each of F3 and B3 primers, 1.6 mM dNTPs (Invitrogen, Waltham, MA, USA), 1 M betaine (Sigma-Aldrich, St. Louis, MO, USA), 4 mM MgSO4 (New England Biolabs, MA, USA), 5 U of AMV reverse transcriptase (Promega), 100 mM dUTP (New England Biolabs), 0.2 U UDG (New England Biolabs), 1× ThermoPol reaction buffer, 8 U of Bst 2.0 warmStart® DNA polymerase (New England Biolabs), and 1× EvaGreen® fluorescent dye (Biotium, Hayward, CA, USA).

    Techniques: Amplification, Lamp Assay