uracil dna glycosylase  (New England Biolabs)


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    Name:
    Uracil DNA Glycosylase UDG
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    Uracil DNA Glycosylase UDG 5 000 units
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    m0280l
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    301
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    5 000 units
    Category:
    DNA Glycosylases
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    Structured Review

    New England Biolabs uracil dna glycosylase
    Uracil DNA Glycosylase UDG
    Uracil DNA Glycosylase UDG 5 000 units
    https://www.bioz.com/result/uracil dna glycosylase/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2020-07
    99/100 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 "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

    3) 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

    4) 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

    5) 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

    6) 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

    7) Product Images from "Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase"

    Article Title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157860

    Glycosylase and lyase activity panel for human NEIL1-WT and the phosphomimetic/ablating mutants. Glycosylase assays were performed by incubating 20 nM of double-stranded DNA substrates (A) Sp:C and (B) AP:C and increasing amounts of enzyme with the following substrate to enzyme ratios: 1:0.5, 1:1, 1:4, and 1:16. “-” indicates a no enzyme negative control. Assays were performed at room temperature for 30 minutes. S and P indicate substrate and product, respectively. Data shown are representative of duplicate experiments.
    Figure Legend Snippet: Glycosylase and lyase activity panel for human NEIL1-WT and the phosphomimetic/ablating mutants. Glycosylase assays were performed by incubating 20 nM of double-stranded DNA substrates (A) Sp:C and (B) AP:C and increasing amounts of enzyme with the following substrate to enzyme ratios: 1:0.5, 1:1, 1:4, and 1:16. “-” indicates a no enzyme negative control. Assays were performed at room temperature for 30 minutes. S and P indicate substrate and product, respectively. Data shown are representative of duplicate experiments.

    Techniques Used: Activity Assay, Negative Control

    Sites of phosphorylation within the NEIL1 DNA glycosylase. (A) Domain map of NEIL1 indicating the position of known sites of phosphorylation. The residues S207, S306, and S61 identified in this study are shown in blue and the Y263 and S269 sites previously identified [ 39 ] are indicated in black. (B) SDS-PAGE gel of SBP-tagged NEIL1 after affinity pull-down from HEK293T cell-extracts overexpressing NEIL1. The gel was stained with Coomassie blue and the NEIL1-SBP band was cut from the gel and digested with trypsin for identification of phosphorylated peptides via LC-MS/MS.
    Figure Legend Snippet: Sites of phosphorylation within the NEIL1 DNA glycosylase. (A) Domain map of NEIL1 indicating the position of known sites of phosphorylation. The residues S207, S306, and S61 identified in this study are shown in blue and the Y263 and S269 sites previously identified [ 39 ] are indicated in black. (B) SDS-PAGE gel of SBP-tagged NEIL1 after affinity pull-down from HEK293T cell-extracts overexpressing NEIL1. The gel was stained with Coomassie blue and the NEIL1-SBP band was cut from the gel and digested with trypsin for identification of phosphorylated peptides via LC-MS/MS.

    Techniques Used: SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    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 "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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    Related Articles

    Concentration Assay:

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

    Incubation:

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

    Formalin-fixed Paraffin-Embedded:

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

    Polymerase Chain Reaction:

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

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

    other:

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

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

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

    Hybridization:

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

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  • 99
    New England Biolabs uracil dna glycosylase
    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil <t>DNA</t> <t>glycosylase</t> (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.
    Uracil Dna Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs escherichia coli uracil dna glycosylase ung
    N <t>-glycosylase</t> activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from <t>DNA</t> by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.
    Escherichia Coli Uracil Dna Glycosylase Ung, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs apobec enzymes
    APOBEC3A is the predominant cytidine deaminase in BRCA cell lines lacking APOBEC3B. (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene ( S2 Table ). (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, <t>A3A</t> shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA <t>APOBEC</t> target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.
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    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.0030135

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

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

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

    N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.

    Journal: Nucleic Acids Research

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    doi:

    Figure Lengend Snippet: N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.

    Article Snippet: Escherichia coli uracil DNA glycosylase (Ung) and exonuclease (Exo) III were purchased from New England Biolabs.

    Techniques: Activity Assay, High Performance Liquid Chromatography, Incubation, Labeling

    APOBEC3A is the predominant cytidine deaminase in BRCA cell lines lacking APOBEC3B. (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene ( S2 Table ). (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.

    Journal: PLoS Genetics

    Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

    doi: 10.1371/journal.pgen.1008545

    Figure Lengend Snippet: APOBEC3A is the predominant cytidine deaminase in BRCA cell lines lacking APOBEC3B. (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene ( S2 Table ). (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.

    Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

    Techniques: Mutagenesis, Expressing, In Vitro, CRISPR, shRNA, Activity Assay, Plasmid Preparation, Incubation, Quantitative RT-PCR, Western Blot

    Correlation of A3A expression with APOBEC-induced mutations in multiple cancer types. RSEM normalized RNA-seq values for A3A (blue) and A3B (red) were obtained for 410 bladder (BLCA), 197 cervical (CESC), and 549 head and neck (HNSC) cancers assessed by the Cancer Genome Atlas. Expression of each APOBEC was normalized to HPRT1 and compared to the minimum estimate of APOBEC-induced mutations in the corresponding samples. A3A expression strongly correlates with mutagenesis in each tumor type by Pearson correlation analysis even when only evaluating APOBEC mutated tumors.

    Journal: PLoS Genetics

    Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

    doi: 10.1371/journal.pgen.1008545

    Figure Lengend Snippet: Correlation of A3A expression with APOBEC-induced mutations in multiple cancer types. RSEM normalized RNA-seq values for A3A (blue) and A3B (red) were obtained for 410 bladder (BLCA), 197 cervical (CESC), and 549 head and neck (HNSC) cancers assessed by the Cancer Genome Atlas. Expression of each APOBEC was normalized to HPRT1 and compared to the minimum estimate of APOBEC-induced mutations in the corresponding samples. A3A expression strongly correlates with mutagenesis in each tumor type by Pearson correlation analysis even when only evaluating APOBEC mutated tumors.

    Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

    Techniques: Expressing, RNA Sequencing Assay, Mutagenesis

    APOBEC3A is the predominant cytidine deaminase in BT474 cells expressing APOBEC3B. (A) The mutation profile of BT474 cells. (B) The number of APOBEC-induced (black bars) and non-APOBEC-induced (grey bars) mutations in BT474 cells. (C) mRNA expression levels of individual APOBEC3 family members relative to HPRT1 expression in BT474 as measured by qRT-PCR. Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean of these measurements. n.d. indicates “not detected.” (D) In vitro cytidine deaminase assay (conducted similarly to Fig 1D and 1E ) of whole-cell extracts generated BT474 cells or BT474 cells transduced with lentiviral vectors to express scramble control, A3A-targeting, or A3B targeting shRNAs. Deaminase reactions were supplemented with either 2 units UGI (NEB #M0281S) or an equal volume of 50% glycerol. Specificity of each shRNA was confirmed by qRT-PCR, and equal protein amounts used in deaminase assays were verified by α-GAPDH western.

    Journal: PLoS Genetics

    Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

    doi: 10.1371/journal.pgen.1008545

    Figure Lengend Snippet: APOBEC3A is the predominant cytidine deaminase in BT474 cells expressing APOBEC3B. (A) The mutation profile of BT474 cells. (B) The number of APOBEC-induced (black bars) and non-APOBEC-induced (grey bars) mutations in BT474 cells. (C) mRNA expression levels of individual APOBEC3 family members relative to HPRT1 expression in BT474 as measured by qRT-PCR. Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean of these measurements. n.d. indicates “not detected.” (D) In vitro cytidine deaminase assay (conducted similarly to Fig 1D and 1E ) of whole-cell extracts generated BT474 cells or BT474 cells transduced with lentiviral vectors to express scramble control, A3A-targeting, or A3B targeting shRNAs. Deaminase reactions were supplemented with either 2 units UGI (NEB #M0281S) or an equal volume of 50% glycerol. Specificity of each shRNA was confirmed by qRT-PCR, and equal protein amounts used in deaminase assays were verified by α-GAPDH western.

    Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

    Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, In Vitro, Generated, Transduction, shRNA, Western Blot

    APOBEC3A expression correlates with the abundance of APOBEC-induced mutations in primary BRCA tumors, despite the similarity between APOBEC3A and APOBEC3B transcripts. (A) The pairwise alignment of the A3A (blue) and A3B (red) transcripts shows a central region of high sequence identity with two unique regions in each transcript. Green, yellow, and red indicate 100%, ≥30%, and

    Journal: PLoS Genetics

    Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

    doi: 10.1371/journal.pgen.1008545

    Figure Lengend Snippet: APOBEC3A expression correlates with the abundance of APOBEC-induced mutations in primary BRCA tumors, despite the similarity between APOBEC3A and APOBEC3B transcripts. (A) The pairwise alignment of the A3A (blue) and A3B (red) transcripts shows a central region of high sequence identity with two unique regions in each transcript. Green, yellow, and red indicate 100%, ≥30%, and

    Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

    Techniques: Expressing, Sequencing

    APOBEC3A mRNA transcript levels correlate with the extent of APOBEC-induced mutations in BRCA cell lines. The average mutation profiles of (A) 14 non-APOBEC-mutated and (B) 14 APOBEC-mutated BRCA cell lines. Specific cell lines in each category are defined in S2 Table . (C) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression was measured by qRT-PCR in non-APOBEC-mutated (N) and APOBEC-mutated (M) BRCA cell lines. Similar results were obtained comparing APOBEC expression to TBP ( S2 Table ). Each circle represents the mean of 3 replicate measurements for an individual cell line. Horizontal bars indicate the median expression for each APOBEC3 family member among the non-APOBEC-mutated or APOBEC-mutated cell lines. Data points corresponding to cell lines without detectable expression of individual APOBECs are not shown on the graph but are included in the calculation of the median. Statistical significance for differences in the expression of a given APOBEC family member between non-APOBEC-mutated and APOBEC-mutated lines was assessed by Mann-Whitney Summed Rank test. ** indicates p = 0.0067. Correlations between A3A expression (blue dots) or A3B expression (red dots) measured by qRT-PCR and the minimum estimate of APOBEC-induced mutations for each of the 28 BRCA cell lines were determined by a Pearson correlation test using mutation lists obtained from (D) the Cancer Cell Line Encyclopedia and (E) the Catalogue of Somatic Mutations in Cancer (COSMIC).

    Journal: PLoS Genetics

    Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

    doi: 10.1371/journal.pgen.1008545

    Figure Lengend Snippet: APOBEC3A mRNA transcript levels correlate with the extent of APOBEC-induced mutations in BRCA cell lines. The average mutation profiles of (A) 14 non-APOBEC-mutated and (B) 14 APOBEC-mutated BRCA cell lines. Specific cell lines in each category are defined in S2 Table . (C) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression was measured by qRT-PCR in non-APOBEC-mutated (N) and APOBEC-mutated (M) BRCA cell lines. Similar results were obtained comparing APOBEC expression to TBP ( S2 Table ). Each circle represents the mean of 3 replicate measurements for an individual cell line. Horizontal bars indicate the median expression for each APOBEC3 family member among the non-APOBEC-mutated or APOBEC-mutated cell lines. Data points corresponding to cell lines without detectable expression of individual APOBECs are not shown on the graph but are included in the calculation of the median. Statistical significance for differences in the expression of a given APOBEC family member between non-APOBEC-mutated and APOBEC-mutated lines was assessed by Mann-Whitney Summed Rank test. ** indicates p = 0.0067. Correlations between A3A expression (blue dots) or A3B expression (red dots) measured by qRT-PCR and the minimum estimate of APOBEC-induced mutations for each of the 28 BRCA cell lines were determined by a Pearson correlation test using mutation lists obtained from (D) the Cancer Cell Line Encyclopedia and (E) the Catalogue of Somatic Mutations in Cancer (COSMIC).

    Article Snippet: Deaminase activity assays with purified APOBEC enzymes (with concentrations A3A or A3B as indicated, 5 units of Uracil DNA glycosylase (NEB), 0.25 μM oligonucleotide substrate, 20 mM Tris HCl pH7.5, 1 mM DTT, and 1 mM EDTA), in a 20 μL volume were incubated for 30 minutes (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9mM EDTA, and 0.0125% SDS) and incubation at 95°C for 10 min. Deaminase activity assays with whole-cell lysates (40 μg of cell lysate, 0.25 μM oligonucleotide substrate, 22.5 mM Tris pH 7.56, 2 mM KCl, 2.5 mM NaCl, 0.005%Triton X-100, 1.5 mM DTT, and 1.25 mM EDTA), in a 20 μL volume were incubated for 24 hrs (unless otherwise indicated) at 37°C and terminated by the addition of 20 μL formamide buffer (47.5% formamide, 9 mM EDTA, and 0.0125% SDS).

    Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, MANN-WHITNEY