uracil dna glycosylase  (New England Biolabs)


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    Uracil DNA Glycosylase UDG
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    Uracil DNA Glycosylase UDG 5 000 units
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    m0280l
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    5 000 units
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    DNA Glycosylases
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    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 91 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2020-08
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    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 "The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1053

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

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

    4) Product Images from "Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries"

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54044-x

    ( A ) Sequence design for the investigation of uracil and abasic site cleavage on microarrays. Each sequence consists of a dT 15 -linker, then a 30mer with either no dUs (control) or an increasing number of dU-incorporations (from 1 to 9) replacing dTs in the following sequence: 5′-TTA CCA TAG AAT CAT GTG CCA TAC ATC ATC-3′. At the 5′-end, a control 25mer is synthesized (QC25), serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). The cleavage process was monitored by recording the hybridization-based fluorescence intensity before and after the UDG-mediated cleavage of uracil nucleotides. ( B ) Small excerpt (ca. 7% of the total synthesis area) of fluorescence scans before and after enzyme exposure. The scans show the fluorescence intensity, resulting from hybridization to a labelled, complementary oligonucleotide. The microarrays were scanned at 5 µm resolution. ( C ) Decrease in fluorescence intensity for the UDG-mediated uracil excision (thus generating abasic sites) as a function of the number of dU nucleotide incorporations per DNA substrate. The actual cleavage efficiencies correlate with the loss of fluorescence intensity resulting from DNA substrate cleavage. The array was incubated for one hour with UDG and the generated abasic sites were subsequently cleaved under alkaline conditions. The decrease in fluorescence intensity was recorded and normalized to the control strand (U0). The normalized intensities, indicated in arbitrary units, were plotted over the number of dUs per DNA substrate. Error bars are SD.
    Figure Legend Snippet: ( A ) Sequence design for the investigation of uracil and abasic site cleavage on microarrays. Each sequence consists of a dT 15 -linker, then a 30mer with either no dUs (control) or an increasing number of dU-incorporations (from 1 to 9) replacing dTs in the following sequence: 5′-TTA CCA TAG AAT CAT GTG CCA TAC ATC ATC-3′. At the 5′-end, a control 25mer is synthesized (QC25), serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). The cleavage process was monitored by recording the hybridization-based fluorescence intensity before and after the UDG-mediated cleavage of uracil nucleotides. ( B ) Small excerpt (ca. 7% of the total synthesis area) of fluorescence scans before and after enzyme exposure. The scans show the fluorescence intensity, resulting from hybridization to a labelled, complementary oligonucleotide. The microarrays were scanned at 5 µm resolution. ( C ) Decrease in fluorescence intensity for the UDG-mediated uracil excision (thus generating abasic sites) as a function of the number of dU nucleotide incorporations per DNA substrate. The actual cleavage efficiencies correlate with the loss of fluorescence intensity resulting from DNA substrate cleavage. The array was incubated for one hour with UDG and the generated abasic sites were subsequently cleaved under alkaline conditions. The decrease in fluorescence intensity was recorded and normalized to the control strand (U0). The normalized intensities, indicated in arbitrary units, were plotted over the number of dUs per DNA substrate. Error bars are SD.

    Techniques Used: Sequencing, Synthesized, Hybridization, Fluorescence, Incubation, Generated

    Schematic illustration of the sequence design for the investigation of E. coli UDG sequence dependences on single- ( A ) and double-stranded DNA substrates. ( B ) In order to investigate the UDG sequence dependence, a single dU is incorporated into a DNA strand and enclosed by 3 permuted bases on each side. A The design for the study of UDG sequence dependence on single-stranded DNA substrates consists of a 15mer dT-linker, a single dU enclosed by 3 permuted bases on each side, followed by a 5′ 25mer sequence (QC25) serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). B For the study of UDG sequence dependence on double-stranded DNA substrates, the sequences were designed to form a hairpin loop. The resulting strands consisted of a 15mer dT-linker, a 11-nt stem, equivalent to the single-stranded design, containing the variable region flanked by dG·dC base pairs, a 4-nt loop followed by the complementary 11nt strand. At the 5′ end, a hybridizable 25mer target sequence (QC25) was synthesized.
    Figure Legend Snippet: Schematic illustration of the sequence design for the investigation of E. coli UDG sequence dependences on single- ( A ) and double-stranded DNA substrates. ( B ) In order to investigate the UDG sequence dependence, a single dU is incorporated into a DNA strand and enclosed by 3 permuted bases on each side. A The design for the study of UDG sequence dependence on single-stranded DNA substrates consists of a 15mer dT-linker, a single dU enclosed by 3 permuted bases on each side, followed by a 5′ 25mer sequence (QC25) serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). B For the study of UDG sequence dependence on double-stranded DNA substrates, the sequences were designed to form a hairpin loop. The resulting strands consisted of a 15mer dT-linker, a 11-nt stem, equivalent to the single-stranded design, containing the variable region flanked by dG·dC base pairs, a 4-nt loop followed by the complementary 11nt strand. At the 5′ end, a hybridizable 25mer target sequence (QC25) was synthesized.

    Techniques Used: Sequencing, Hybridization, Synthesized

    (Left) Schematic illustration of the UDG-mediated generation of single nucleotide gaps on nucleic acid strands. In a first step, UDG catalyzes the excision of uracil, leading to the formation of an abasic site. This AP-site can then either be cleaved by the lyase activity of specific endonucleases, or chemically. The USER enzyme, a mixture of UDG and Endonuclease VIII, combines AP-site formation and cleavage in a single solution. (Right) Molecular structures indicating the generation of a single nucleotide gap/strand cleavage via a β- and a subsequent δ-elimination reaction. First, UDG hydrolyzes the glycosidic bond from the uracil-containing DNA strand. The ribose at the apyrimidinic site lacks a glycosidic bond and is therefore highly unstable and converts rapidly into its reactive open-chain aldehyde, its hemiacetal or its hydrate form. The lyase activity of AP-endonucleases, or the exposure to either basic or acidic conditions, initiates a β-elimination reaction, resulting in the cleavage of the phosphodiester backbone 3′ to the AP-site and the formation of an α,β-unsaturated aldehyde. Subsequent δ-elimination induces DNA strand cleavage 5′ to the AP-site resulting in the generation of a single-nucleotide gap in dsDNA or strand cleavage in ssDNA.
    Figure Legend Snippet: (Left) Schematic illustration of the UDG-mediated generation of single nucleotide gaps on nucleic acid strands. In a first step, UDG catalyzes the excision of uracil, leading to the formation of an abasic site. This AP-site can then either be cleaved by the lyase activity of specific endonucleases, or chemically. The USER enzyme, a mixture of UDG and Endonuclease VIII, combines AP-site formation and cleavage in a single solution. (Right) Molecular structures indicating the generation of a single nucleotide gap/strand cleavage via a β- and a subsequent δ-elimination reaction. First, UDG hydrolyzes the glycosidic bond from the uracil-containing DNA strand. The ribose at the apyrimidinic site lacks a glycosidic bond and is therefore highly unstable and converts rapidly into its reactive open-chain aldehyde, its hemiacetal or its hydrate form. The lyase activity of AP-endonucleases, or the exposure to either basic or acidic conditions, initiates a β-elimination reaction, resulting in the cleavage of the phosphodiester backbone 3′ to the AP-site and the formation of an α,β-unsaturated aldehyde. Subsequent δ-elimination induces DNA strand cleavage 5′ to the AP-site resulting in the generation of a single-nucleotide gap in dsDNA or strand cleavage in ssDNA.

    Techniques Used: Activity Assay

    Representative sequence motifs for the UDG-mediated uracil cleavage on double- ( A , B ) and single-stranded ( C , D ) DNA strands. Substrates were incubated with UDG for different time periods ranging from 5 seconds to 30 minutes (since the cleavage motifs showed little to no sequence dependence, only the 5s, 30s, 60s and 120s were determined for ssDNA). The sequence motifs were extracted from the 1% (41 of 4096 sequences) most cleaved ( A , C ) and least cleaved ( B , D ) sequences of the library.
    Figure Legend Snippet: Representative sequence motifs for the UDG-mediated uracil cleavage on double- ( A , B ) and single-stranded ( C , D ) DNA strands. Substrates were incubated with UDG for different time periods ranging from 5 seconds to 30 minutes (since the cleavage motifs showed little to no sequence dependence, only the 5s, 30s, 60s and 120s were determined for ssDNA). The sequence motifs were extracted from the 1% (41 of 4096 sequences) most cleaved ( A , C ) and least cleaved ( B , D ) sequences of the library.

    Techniques Used: Sequencing, Incubation

    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 "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

    7) Product Images from "Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase"

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase

    Journal: Oncotarget

    doi:

    Detection of true KRAS and EGFR mutations after UDG treatment The effect of UDG treatment on detection of various types of true mutations are examined using a set of FFPE DNA samples harbouring either KRAS or EGFR exon 19 deletions and exon 20 insertion mutations. All KRAS -mutant and EGFR -mutant samples are correctly identifiable by HRM or Sanger sequencing regardless of UDG treatment. The positions of KRAS mutations and representative nucleotides of EGFR mutations are indicated by a red asterisk. Panel A: Sequence traces of KRAS exon 2 before and after UDG treatment. Both TX23 and TX63 samples harbour KRAS c.35G > A mutations and HCT116 cell line DNA contains a KRAS c.38G > A mutation. Panel B: Sequence traces of EGFR exon 19 before and after UDG treatment. Both TX35 and H1650 harbour EGFR p.E746_A750del mutations and TX48 harbours a p.T751_I759delinsN mutation. Panel C: Sequence traces of EGFR exon 20 before and after UDG treatment. TX202, TX383 and TX440 samples harbour EGFR p.C775_R776insPA, p.H773_R776insYNPY, and p.D770_H773insGSVD, respectively. Panel D: Difference plots of low-level KRAS- mutant samples before (left) and after UDG treatment (right). KRAS mutations detected are c.35G > T (N1 9), c.35G > T (N1 46), c.35G > C (N1 53), c.34G > T (TX450). RPMI8226 cell line DNA contains a KRAS c.35G > C mutation.
    Figure Legend Snippet: Detection of true KRAS and EGFR mutations after UDG treatment The effect of UDG treatment on detection of various types of true mutations are examined using a set of FFPE DNA samples harbouring either KRAS or EGFR exon 19 deletions and exon 20 insertion mutations. All KRAS -mutant and EGFR -mutant samples are correctly identifiable by HRM or Sanger sequencing regardless of UDG treatment. The positions of KRAS mutations and representative nucleotides of EGFR mutations are indicated by a red asterisk. Panel A: Sequence traces of KRAS exon 2 before and after UDG treatment. Both TX23 and TX63 samples harbour KRAS c.35G > A mutations and HCT116 cell line DNA contains a KRAS c.38G > A mutation. Panel B: Sequence traces of EGFR exon 19 before and after UDG treatment. Both TX35 and H1650 harbour EGFR p.E746_A750del mutations and TX48 harbours a p.T751_I759delinsN mutation. Panel C: Sequence traces of EGFR exon 20 before and after UDG treatment. TX202, TX383 and TX440 samples harbour EGFR p.C775_R776insPA, p.H773_R776insYNPY, and p.D770_H773insGSVD, respectively. Panel D: Difference plots of low-level KRAS- mutant samples before (left) and after UDG treatment (right). KRAS mutations detected are c.35G > T (N1 9), c.35G > T (N1 46), c.35G > C (N1 53), c.34G > T (TX450). RPMI8226 cell line DNA contains a KRAS c.35G > C mutation.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Mutagenesis, Sequencing

    The effect of UDG treatment on sequence artefacts in AKT1 as assessed using LCN-HRM The frequency of sequence artefacts in the AKT1 sequence were assessed in three FFPE DNA samples (SCC7, SCC8, and SCC14) with and without UDG treatment using LCN-HRM. The melting profiles of 60 individual LCN-HRM products are presented in the negative first derivative plot. Positive LCN-HRM reactions are shown in red and wild-type reactions are shown in green. There is a marked reduction in the number of LCN-HRM positive reactions after UDG treatment in all three samples. In SCC7, a total of 34 reactions were positive without UDG treatment (Panel A), which is markedly reduced to 5 after UDG treatment (Panel B). In SCC8, 24 and 10 LCN-HRM reactions were positive without (Panel C) and with UDG treatment (Panel D), and 20 and 3 LCN-HRM positives are found without (Panel E) and with UDG treatment (Panel F) in SCC14.
    Figure Legend Snippet: The effect of UDG treatment on sequence artefacts in AKT1 as assessed using LCN-HRM The frequency of sequence artefacts in the AKT1 sequence were assessed in three FFPE DNA samples (SCC7, SCC8, and SCC14) with and without UDG treatment using LCN-HRM. The melting profiles of 60 individual LCN-HRM products are presented in the negative first derivative plot. Positive LCN-HRM reactions are shown in red and wild-type reactions are shown in green. There is a marked reduction in the number of LCN-HRM positive reactions after UDG treatment in all three samples. In SCC7, a total of 34 reactions were positive without UDG treatment (Panel A), which is markedly reduced to 5 after UDG treatment (Panel B). In SCC8, 24 and 10 LCN-HRM reactions were positive without (Panel C) and with UDG treatment (Panel D), and 20 and 3 LCN-HRM positives are found without (Panel E) and with UDG treatment (Panel F) in SCC14.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

    Sequence artefacts detected in FFPE DNA by Sanger sequencing Multiple non-reproducible sequence artefacts detected in the AKT1 sequence from FFPE DNA are shown. Panel A: Four sequence artefacts detected in the SCC8 sample without UDG treatment. Three of the sequence artefacts (c.81C > T, c.145G > A and c.153C > T) were found in the same amplicon from one replicate and the c.110G > A change was detected in the second replicate. Panel B: Four sequence artefacts detected in three FFPE DNA samples (SCC7, SCC11, and SCC14) after UDG treatment. c.122G > A and c.143G > A changes were detected in different replicates from the SCC7 sample. A c.125C > T (SCC11) and a c.175C > T (SCC14) change was found in a replicate of SCC11 and SCC14 respectively. All of the C:G > T:A changes that were found after UDG treatment were detected in the sequence context of CpG dinucleotides.
    Figure Legend Snippet: Sequence artefacts detected in FFPE DNA by Sanger sequencing Multiple non-reproducible sequence artefacts detected in the AKT1 sequence from FFPE DNA are shown. Panel A: Four sequence artefacts detected in the SCC8 sample without UDG treatment. Three of the sequence artefacts (c.81C > T, c.145G > A and c.153C > T) were found in the same amplicon from one replicate and the c.110G > A change was detected in the second replicate. Panel B: Four sequence artefacts detected in three FFPE DNA samples (SCC7, SCC11, and SCC14) after UDG treatment. c.122G > A and c.143G > A changes were detected in different replicates from the SCC7 sample. A c.125C > T (SCC11) and a c.175C > T (SCC14) change was found in a replicate of SCC11 and SCC14 respectively. All of the C:G > T:A changes that were found after UDG treatment were detected in the sequence context of CpG dinucleotides.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded, Amplification

    UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.
    Figure Legend Snippet: UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Amplification

    Uracil lesions in FFPE DNA leading to sequence artefacts and in vitro removal of uracil by uracil-DNA glycosylase Spontaneous cytosine deamination is a frequent DNA damage that takes place at a rate of 70 - 200 events per day in the human genome. In normal cells, the resulting uracil lesions are effectively removed by UDG. The resulting abasic sites are then repaired by the base excision DNA repair system. However, in biopsy specimen, if cytosine deamination occurs during sample collection, formalin fixation, and fixed tissue storage, the resulting uracil lesions cannot be repaired due to the absence of functional DNA repair proteins. When DNA is extracted from the tissue with uracil lesions and then used as template for PCR amplification, transitional C:G > T:A sequence artefacts are generated as uracil efficiently pairs with adenine. The generation of artefactual C:G > T:A transitions from the uracil lesions in FFPE DNA can be effectively eliminated by treating FFPE DNA with UDG in vitro prior to PCR amplification. Abasic sites generated by the removal of uracil bases may reduce the extension by DNA polymerase and strand breakage during the repetitive exposure to high temperature during PCR cycling. Thus, treatment of FFPE DNA with UDG prior to PCR amplification eliminates the generation of artefactual C:G > T:A transitions arising from uracil lesions.
    Figure Legend Snippet: Uracil lesions in FFPE DNA leading to sequence artefacts and in vitro removal of uracil by uracil-DNA glycosylase Spontaneous cytosine deamination is a frequent DNA damage that takes place at a rate of 70 - 200 events per day in the human genome. In normal cells, the resulting uracil lesions are effectively removed by UDG. The resulting abasic sites are then repaired by the base excision DNA repair system. However, in biopsy specimen, if cytosine deamination occurs during sample collection, formalin fixation, and fixed tissue storage, the resulting uracil lesions cannot be repaired due to the absence of functional DNA repair proteins. When DNA is extracted from the tissue with uracil lesions and then used as template for PCR amplification, transitional C:G > T:A sequence artefacts are generated as uracil efficiently pairs with adenine. The generation of artefactual C:G > T:A transitions from the uracil lesions in FFPE DNA can be effectively eliminated by treating FFPE DNA with UDG in vitro prior to PCR amplification. Abasic sites generated by the removal of uracil bases may reduce the extension by DNA polymerase and strand breakage during the repetitive exposure to high temperature during PCR cycling. Thus, treatment of FFPE DNA with UDG prior to PCR amplification eliminates the generation of artefactual C:G > T:A transitions arising from uracil lesions.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sequencing, In Vitro, Functional Assay, Polymerase Chain Reaction, Amplification, Generated

    The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.
    Figure Legend Snippet: The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.

    Techniques Used: Formalin-fixed Paraffin-Embedded, HRM Assay, Polymerase Chain Reaction, Amplification, Concentration Assay

    8) Product Images from "Expression and purification of active mouse and human NEIL3 proteins"

    Article Title: Expression and purification of active mouse and human NEIL3 proteins

    Journal: Protein Expression and Purification

    doi: 10.1016/j.pep.2012.04.022

    DNA glycosylase/lyase activity of MmuNeil3Δ324 and NEIL3Δ324. Single- and double-stranded substrates containing Tg, Sp, or an AP site (25 nM) were incubated with 25 nM active MmuNeil3Δ324 (lane 4) and NEIL3Δ324 (lane 5),
    Figure Legend Snippet: DNA glycosylase/lyase activity of MmuNeil3Δ324 and NEIL3Δ324. Single- and double-stranded substrates containing Tg, Sp, or an AP site (25 nM) were incubated with 25 nM active MmuNeil3Δ324 (lane 4) and NEIL3Δ324 (lane 5),

    Techniques Used: Activity Assay, Incubation

    9) Product Images from "Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair"

    Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2019.02.001

    BER activity in MEFs. Linear oligonucleotide duplexes containing a single uracil (A) or F lesion (B) were used as substrates in repair assays with WT and POLB −/− . Where indicated (* time points), after the reaction an aliquot was treated with UDG and Ape1 (U substrate) or Ape1 alone (F-substrate) to cleave any unrepaired DNA. After electrophoresis, the resolved products from the UDG/Ape1-treated treated samples were quantified to determine the fraction of DNA repaired. Positions of the substrate (S), intermediates, and repaired product (P) bands are indicated. For each experiment, a representative gel image is shown. The means ± standard errors are plotted (right; n=3).
    Figure Legend Snippet: BER activity in MEFs. Linear oligonucleotide duplexes containing a single uracil (A) or F lesion (B) were used as substrates in repair assays with WT and POLB −/− . Where indicated (* time points), after the reaction an aliquot was treated with UDG and Ape1 (U substrate) or Ape1 alone (F-substrate) to cleave any unrepaired DNA. After electrophoresis, the resolved products from the UDG/Ape1-treated treated samples were quantified to determine the fraction of DNA repaired. Positions of the substrate (S), intermediates, and repaired product (P) bands are indicated. For each experiment, a representative gel image is shown. The means ± standard errors are plotted (right; n=3).

    Techniques Used: Activity Assay, Electrophoresis

    Analysis of repaired plasmid DNA recovered after transfection. Recovered DNA (A, lane 2) was confirmed by restriction digestion with AatII and BamHI (A, lane 3) . The pGEM3Zf (+) plasmid was loaded as a marker (A, lane1) . Leftmost lane: a 1-kb ladder. The amount of DNA repaired was estimated by treating the samples with UDG and Ape1 (using amounts sufficient to cleave all unrepaired DNA, as determined by experiments with the unrepaired substrate), and the products were resolved on a 0.8% agarose gel containing ethidium bromide. Analysis of the DNA recovered from two experiments is shown (B, lane 1 and 2) .
    Figure Legend Snippet: Analysis of repaired plasmid DNA recovered after transfection. Recovered DNA (A, lane 2) was confirmed by restriction digestion with AatII and BamHI (A, lane 3) . The pGEM3Zf (+) plasmid was loaded as a marker (A, lane1) . Leftmost lane: a 1-kb ladder. The amount of DNA repaired was estimated by treating the samples with UDG and Ape1 (using amounts sufficient to cleave all unrepaired DNA, as determined by experiments with the unrepaired substrate), and the products were resolved on a 0.8% agarose gel containing ethidium bromide. Analysis of the DNA recovered from two experiments is shown (B, lane 1 and 2) .

    Techniques Used: Plasmid Preparation, Transfection, Marker, Agarose Gel Electrophoresis

    Uracil-repair patch size distribution. ( A ) Quantitative analysis of total repair of the circular substrate using MEF extracts. Reaction mixtures containing 0.1 pmol of circular substrate in the standard in vitro ) and 3.5 μg of cell extract were incubated at 37°C for 60 min, followed by incubation at 65°C for 20 min to inactivate enzymes. The total repaired DNA in each sample was determined by adding UDG and Ape1 to ensure cleavage of any unrepaired DNA. Reaction products were resolved on a 0.8% agarose gel and quantified using ImageJ. ( B .
    Figure Legend Snippet: Uracil-repair patch size distribution. ( A ) Quantitative analysis of total repair of the circular substrate using MEF extracts. Reaction mixtures containing 0.1 pmol of circular substrate in the standard in vitro ) and 3.5 μg of cell extract were incubated at 37°C for 60 min, followed by incubation at 65°C for 20 min to inactivate enzymes. The total repaired DNA in each sample was determined by adding UDG and Ape1 to ensure cleavage of any unrepaired DNA. Reaction products were resolved on a 0.8% agarose gel and quantified using ImageJ. ( B .

    Techniques Used: In Vitro, Incubation, Agarose Gel Electrophoresis

    10) Product Images from "Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies"

    Article Title: Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195048

    Structure of Pot1A3G CTD with ssDNA. A) Schematic of the Pot1A3G CTD fusion protein design. Pot1 (pink) is fused directly to the N-terminus of A3G CTD (blue). The ssDNA contains both Pot1 and A3G binding sites: the Pot1 site in dark gray and the A3G hotspot in light gray with the linker sequence in smaller font. The resolved adenine in the -1 pocket is colored orange and the expected deaminated cytidine is blue. B) Size exclusion binding test shows that Pot1A3G CTD binds to the ssDNA substrate. Pot1A3G CTD alone is in black, the ssDNA is in gray, and the mixture of the two is in red. C) Deamination activity using a UDG-dependent cleavage assay. The Pot1A3G CTD fusion protein has the same deamination activity as that of A3G CTD . D) Schematic and structure of the Pot1A3G CTD in complex with DNA as observed in the crystal. The dA nucleotide bound to the -1 pocket is shown in orange. Two copies of the complex observed in the asymmetric unit are shown in blue (A3G), pink (Pot1), and grey/orange (DNA). The red star (schematic) and red sphere (structure) represent the zinc ion found in the catalytic site. The inset shows the 2Fo-Fc density (1σ contour level) observed for the adenine in the -1 nucleotide-binding pocket.
    Figure Legend Snippet: Structure of Pot1A3G CTD with ssDNA. A) Schematic of the Pot1A3G CTD fusion protein design. Pot1 (pink) is fused directly to the N-terminus of A3G CTD (blue). The ssDNA contains both Pot1 and A3G binding sites: the Pot1 site in dark gray and the A3G hotspot in light gray with the linker sequence in smaller font. The resolved adenine in the -1 pocket is colored orange and the expected deaminated cytidine is blue. B) Size exclusion binding test shows that Pot1A3G CTD binds to the ssDNA substrate. Pot1A3G CTD alone is in black, the ssDNA is in gray, and the mixture of the two is in red. C) Deamination activity using a UDG-dependent cleavage assay. The Pot1A3G CTD fusion protein has the same deamination activity as that of A3G CTD . D) Schematic and structure of the Pot1A3G CTD in complex with DNA as observed in the crystal. The dA nucleotide bound to the -1 pocket is shown in orange. Two copies of the complex observed in the asymmetric unit are shown in blue (A3G), pink (Pot1), and grey/orange (DNA). The red star (schematic) and red sphere (structure) represent the zinc ion found in the catalytic site. The inset shows the 2Fo-Fc density (1σ contour level) observed for the adenine in the -1 nucleotide-binding pocket.

    Techniques Used: Binding Assay, Sequencing, Activity Assay, Cleavage Assay

    11) 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

    12) 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

    13) Product Images from "Base Excision Repair in Early Zebrafish Development: Evidence for DNA Polymerase Switching and Standby AP endonuclease Activity"

    Article Title: Base Excision Repair in Early Zebrafish Development: Evidence for DNA Polymerase Switching and Standby AP endonuclease Activity

    Journal: Biochemistry

    doi: 10.1021/bi900253d

    Uracil removal is performed by uracil DNA glycosylase, as shown by complete sensitivity to the UDG inhibitor Ugi
    Figure Legend Snippet: Uracil removal is performed by uracil DNA glycosylase, as shown by complete sensitivity to the UDG inhibitor Ugi

    Techniques Used:

    14) Product Images from "Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells"

    Article Title: Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.1.159-166.2003

    Fluorescence assay of uracil DNA glycosylase activity. RK13 cells were mock infected or infected with recombinant virus at 10 PFU/cell. After 8 h at 37°C, the cells were harvested and lysed. Fluorescence assays were performed using 5 μg of cell extract protein and 1 μg of supercoiled pUC-19 DNA containing (A and B) or lacking (C and D) uracil residues. At the indicated times, aliquots of the reaction mixture were removed into pH 12 buffer, and fluorescence was measured before and after the mixtures were boiled. The percent fluorescence represents the amount of supercoiled pUC-19 remaining. The presence of nicks in the uracil containing pUC 19, prepared from E. coli CJ236 ( dut ung ) cells, accounted for the values of
    Figure Legend Snippet: Fluorescence assay of uracil DNA glycosylase activity. RK13 cells were mock infected or infected with recombinant virus at 10 PFU/cell. After 8 h at 37°C, the cells were harvested and lysed. Fluorescence assays were performed using 5 μg of cell extract protein and 1 μg of supercoiled pUC-19 DNA containing (A and B) or lacking (C and D) uracil residues. At the indicated times, aliquots of the reaction mixture were removed into pH 12 buffer, and fluorescence was measured before and after the mixtures were boiled. The percent fluorescence represents the amount of supercoiled pUC-19 remaining. The presence of nicks in the uracil containing pUC 19, prepared from E. coli CJ236 ( dut ung ) cells, accounted for the values of

    Techniques Used: Fluorescence, Activity Assay, Infection, Recombinant

    Sequence alignment of uracil DNA glycosylase proteins of viruses and cells. The shaded segments indicate identical amino acids. The circles indicate conserved active-site residues. The solid circles indicate the conserved amino acids of the vaccinia virus uracil DNA glycosylase that were mutated in this study. The numbers of the amino acid residues of each protein are indicated at the left and right of the lines. HSV-1, herpes simplex virus type 1; VAC, vaccinia virus; MPXV, monkeypox virus; VAR, variola major virus; EVM, ectromelia virus; CMPV, camelpox virus; CPXV, cowpox virus; YAB, Yaba-like disease virus; SWPV, swinepox virus; MYX, myxoma virus; RFB, rabbit fibroma virus; LSD, lumpy skin disease virus; SPPV, sheeppox virus; MCV, molluscum contagiosum virus; FPV, fowlpox virus.
    Figure Legend Snippet: Sequence alignment of uracil DNA glycosylase proteins of viruses and cells. The shaded segments indicate identical amino acids. The circles indicate conserved active-site residues. The solid circles indicate the conserved amino acids of the vaccinia virus uracil DNA glycosylase that were mutated in this study. The numbers of the amino acid residues of each protein are indicated at the left and right of the lines. HSV-1, herpes simplex virus type 1; VAC, vaccinia virus; MPXV, monkeypox virus; VAR, variola major virus; EVM, ectromelia virus; CMPV, camelpox virus; CPXV, cowpox virus; YAB, Yaba-like disease virus; SWPV, swinepox virus; MYX, myxoma virus; RFB, rabbit fibroma virus; LSD, lumpy skin disease virus; SPPV, sheeppox virus; MCV, molluscum contagiosum virus; FPV, fowlpox virus.

    Techniques Used: Sequencing

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) Product Images from "Characterization of a Thermostable 8-Oxoguanine DNA Glycosylase Specific for GO/N Mismatches from the Thermoacidophilic Archaeon Thermoplasma volcanium"

    Article Title: Characterization of a Thermostable 8-Oxoguanine DNA Glycosylase Specific for GO/N Mismatches from the Thermoacidophilic Archaeon Thermoplasma volcanium

    Journal: Archaea

    doi: 10.1155/2016/8734894

    GO glycosylase/lyase activity assay of putative TvoOgg. (a) Time course of TvoOgg glycosylase activity on substrate ∗ GO/C. 34-bp heteroduplex DNA containing a ∗ GO/C mismatch was incubated with (lanes 2 to 7) or without (lane 1) 1 pmol of TvoOgg. The uncut 34-mer DNA substrate (S) and cleaved 13-mer product (P) are indicated on the left. (b) Dose dependency of ∗ GO/C mismatch-specific DNA glycosylase activity of TvoOgg on 34-bp double-stranded DNA containing GO/C. The uncut 34-mer DNA substrates (S) and cleaved 13-mer products (P) are indicated on the left. Asterisk indicated the 5′FAM-labeled strand.
    Figure Legend Snippet: GO glycosylase/lyase activity assay of putative TvoOgg. (a) Time course of TvoOgg glycosylase activity on substrate ∗ GO/C. 34-bp heteroduplex DNA containing a ∗ GO/C mismatch was incubated with (lanes 2 to 7) or without (lane 1) 1 pmol of TvoOgg. The uncut 34-mer DNA substrate (S) and cleaved 13-mer product (P) are indicated on the left. (b) Dose dependency of ∗ GO/C mismatch-specific DNA glycosylase activity of TvoOgg on 34-bp double-stranded DNA containing GO/C. The uncut 34-mer DNA substrates (S) and cleaved 13-mer products (P) are indicated on the left. Asterisk indicated the 5′FAM-labeled strand.

    Techniques Used: Activity Assay, Incubation, Labeling

    Requirement of GO for the substrates of TvoOgg. TvoOgg glycosylase/lyase activity was determined by analysis of the products in the presence (+) or absence (−) of 50 nM of TvoOgg on 34-bp double-stranded DNA containing (a) ∗ GO/N, (b) GO/N ∗ , (c) ∗ U/N, or (d) A/N ∗ (N means A, T, G, or C). The uncut 34-mer DNA substrates (S) and cleaved 13-mer products (P) are indicated on the left. Asterisks indicate 5′-FAM-labeled strand.
    Figure Legend Snippet: Requirement of GO for the substrates of TvoOgg. TvoOgg glycosylase/lyase activity was determined by analysis of the products in the presence (+) or absence (−) of 50 nM of TvoOgg on 34-bp double-stranded DNA containing (a) ∗ GO/N, (b) GO/N ∗ , (c) ∗ U/N, or (d) A/N ∗ (N means A, T, G, or C). The uncut 34-mer DNA substrates (S) and cleaved 13-mer products (P) are indicated on the left. Asterisks indicate 5′-FAM-labeled strand.

    Techniques Used: Activity Assay, Labeling

    Temperature dependency of GO glycosylase activity (a), or AP lyase activity (b), in the presence (+) or absence (−) of 1 pmol of TvoOgg with a 34-bp heteroduplex DNA containing GO/C (a) or AP/C (b), respectively, mismatched at different temperatures for 30 min. The strand containing GO and AP was 5′-end labeled with FAM. The uncut 34-mer DNA substrate (S) and cleaved 13-mer product (P) were indicated on the left. The signal intensities of each product were measured and quantified with Pharos FX. Data are presented as the mean ± SD of three independent measurements. Asterisks indicate the 5′-end labeled strand.
    Figure Legend Snippet: Temperature dependency of GO glycosylase activity (a), or AP lyase activity (b), in the presence (+) or absence (−) of 1 pmol of TvoOgg with a 34-bp heteroduplex DNA containing GO/C (a) or AP/C (b), respectively, mismatched at different temperatures for 30 min. The strand containing GO and AP was 5′-end labeled with FAM. The uncut 34-mer DNA substrate (S) and cleaved 13-mer product (P) were indicated on the left. The signal intensities of each product were measured and quantified with Pharos FX. Data are presented as the mean ± SD of three independent measurements. Asterisks indicate the 5′-end labeled strand.

    Techniques Used: Activity Assay, Labeling

    Amino acid sequence alignments of the TVG_RS00315 protein (TvoOgg; WP_010916318.1) with 8-oxoguanine DNA glycosylase of Methanocaldococcus jannaschii (MjaOgg; Q58134) and 8-oxoguanine DNA glycosylase of Sulfolobus solfataricus (SsoOgg; WP_009992328). The amino acid residues in bold are conserved between the three proteins. “H” and “h” indicate alpha helices and hairpin structures, respectively. The catalytic residue of a conserved aspartate is boxed. Asterisks, colons, and dots indicate positions which have fully conserved, strongly similar, and weakly similar residues, respectively.
    Figure Legend Snippet: Amino acid sequence alignments of the TVG_RS00315 protein (TvoOgg; WP_010916318.1) with 8-oxoguanine DNA glycosylase of Methanocaldococcus jannaschii (MjaOgg; Q58134) and 8-oxoguanine DNA glycosylase of Sulfolobus solfataricus (SsoOgg; WP_009992328). The amino acid residues in bold are conserved between the three proteins. “H” and “h” indicate alpha helices and hairpin structures, respectively. The catalytic residue of a conserved aspartate is boxed. Asterisks, colons, and dots indicate positions which have fully conserved, strongly similar, and weakly similar residues, respectively.

    Techniques Used: Sequencing

    21) Product Images from "Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations"

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    Journal: eLife

    doi: 10.7554/eLife.51605

    Purification and activity of NEIL2. ( A ) Purified NEIL2-His 6 (50 ng) from E. coli was subjected to NuPAGE and stained with Coomassie blue. ( B ) Activity of purified NEIL2-His 6 on 5’-[ 32 P]-labeled oligonucleotides (51 nt) containing: hydroxyuracil (OHU), OHU/G, U, or U/G. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. S, substrate; P, product. ( C ) Validation of UDG-generated AP sites from 5’-[ 32 P]-labeled single-stranded and double-stranded oligonucleotides (35 nt) by treatment with NaOH. AP sites are lysed by alkali treatment. ( D ) NEIL2 cleaves Fluorescein (Fluor)-labeled U-containing single strand oligonucleotide (39 nt) in the presence of UDG. S, substrate; P, product.
    Figure Legend Snippet: Purification and activity of NEIL2. ( A ) Purified NEIL2-His 6 (50 ng) from E. coli was subjected to NuPAGE and stained with Coomassie blue. ( B ) Activity of purified NEIL2-His 6 on 5’-[ 32 P]-labeled oligonucleotides (51 nt) containing: hydroxyuracil (OHU), OHU/G, U, or U/G. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. S, substrate; P, product. ( C ) Validation of UDG-generated AP sites from 5’-[ 32 P]-labeled single-stranded and double-stranded oligonucleotides (35 nt) by treatment with NaOH. AP sites are lysed by alkali treatment. ( D ) NEIL2 cleaves Fluorescein (Fluor)-labeled U-containing single strand oligonucleotide (39 nt) in the presence of UDG. S, substrate; P, product.

    Techniques Used: Purification, Activity Assay, Staining, Labeling, Generated

    Reaction mechanisms for the APE1 and NEIL2-PNKP pathways. Glycosylase UDG removes U and generates an AP site on U/G mismatch (only top strand is shown). For the APE1 pathway, APE1 hydrolyzes the deoxyribose-phosphate backbone and generates a 5’-deoxyribophosphate (5’dRP) and a 3’-hydroxyl (3’OH). DNA Polymerase β (Polβ) adds a C to the 3’OH and removes the 5’dRP. For the NEIL2-PNKP pathway, NEIL2 catalyzes β,δ-elimination step by step at the AP site and generates a 5’-phosphate (5’P) and a 3’P. PNKP phosphatase hydrolyzes 3’P to generate a 3’OH primer for Polβ.
    Figure Legend Snippet: Reaction mechanisms for the APE1 and NEIL2-PNKP pathways. Glycosylase UDG removes U and generates an AP site on U/G mismatch (only top strand is shown). For the APE1 pathway, APE1 hydrolyzes the deoxyribose-phosphate backbone and generates a 5’-deoxyribophosphate (5’dRP) and a 3’-hydroxyl (3’OH). DNA Polymerase β (Polβ) adds a C to the 3’OH and removes the 5’dRP. For the NEIL2-PNKP pathway, NEIL2 catalyzes β,δ-elimination step by step at the AP site and generates a 5’-phosphate (5’P) and a 3’P. PNKP phosphatase hydrolyzes 3’P to generate a 3’OH primer for Polβ.

    Techniques Used:

    22) Product Images from "Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair"

    Article Title: Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw054

    Bsu LigD is endowed with an AP lyase activity. ( A ) Analysis of the capacity of BsuL igD to incise an internal natural abasic site. The [α 32 P]3′-labeled 2′-deoxyuridine-containing substrate was treated with 27 nM E. coli UDG (lane c ), leaving an intact AP site. The resulting AP-containing DNA was incubated in the presence of either 5 nM h APE1 that cleaves 5′ to the AP site, 3.5 nM EndoIII that incises 3′ to the AP site, or increasing concentrations of Bsu LigD (0, 29, 57 and 114 nM) for 1 h at 30°C, as described in Materials and Methods. After incubation samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. ( B ) Analysis of the capacity of Bsu LigD to incise an internal tetrahydrofuran (H). The 3′ [α 32 P]3′-dAMP labeled oligonucleotide containing the lyase-resistant analogue tetrahydrofuran (H) was incubated in the presence of either h APE1, EndoIII or increasing concentrations of Bsu LigD as described above. Position corresponding to the products 16-mer 5′-dRP and 16-mer 5′-P is indicated. The figure is a composite image made from different parts of the same experiment.
    Figure Legend Snippet: Bsu LigD is endowed with an AP lyase activity. ( A ) Analysis of the capacity of BsuL igD to incise an internal natural abasic site. The [α 32 P]3′-labeled 2′-deoxyuridine-containing substrate was treated with 27 nM E. coli UDG (lane c ), leaving an intact AP site. The resulting AP-containing DNA was incubated in the presence of either 5 nM h APE1 that cleaves 5′ to the AP site, 3.5 nM EndoIII that incises 3′ to the AP site, or increasing concentrations of Bsu LigD (0, 29, 57 and 114 nM) for 1 h at 30°C, as described in Materials and Methods. After incubation samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. ( B ) Analysis of the capacity of Bsu LigD to incise an internal tetrahydrofuran (H). The 3′ [α 32 P]3′-dAMP labeled oligonucleotide containing the lyase-resistant analogue tetrahydrofuran (H) was incubated in the presence of either h APE1, EndoIII or increasing concentrations of Bsu LigD as described above. Position corresponding to the products 16-mer 5′-dRP and 16-mer 5′-P is indicated. The figure is a composite image made from different parts of the same experiment.

    Techniques Used: Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    Formation of Bsu LigD-DNA adducts. ( A ) Dependence of Bsu LigD-DNA cross-link on NaBH 4 . Reactions were performed as described in Materials and Methods, incubating 95 nM Bsu LigD with 2.6 nM of the 3′ [α 32 P]3′-dAMP labeled DNA substrate depicted on top of the figure, in the presence of 10 μM CTP, 0.64 mM MnCl 2 and either 100 mM NaBH 4 or NaCl (as indicated). Left panel: Coomassie blue staining after SDS–PAGE of purified Bsu LigD. Right panel: autoradiography of corresponding protein-DNA adducts after the SDS–PAGE separation shown in left panel. When indicated, protein was previously incubated with 0.05 U of thrombin at 20°C for 60 min. ( B ) Adduct formation is dependent on the presence of an abasic site. Reactions were performed as in described in (A) but using as substrate 3.6 nM of the 3′ [α 32 P]3′-dAMP labeled oligonucleotide without removing the uracil ( absence of AP site ) or after treatment with E. coli UDG ( presence of AP site ), in the presence of either 100 mM NaBH 4 or NaCl (as indicated). Autoradiography of corresponding protein-DNA adduct after the SDS–PAGE separation is shown.
    Figure Legend Snippet: Formation of Bsu LigD-DNA adducts. ( A ) Dependence of Bsu LigD-DNA cross-link on NaBH 4 . Reactions were performed as described in Materials and Methods, incubating 95 nM Bsu LigD with 2.6 nM of the 3′ [α 32 P]3′-dAMP labeled DNA substrate depicted on top of the figure, in the presence of 10 μM CTP, 0.64 mM MnCl 2 and either 100 mM NaBH 4 or NaCl (as indicated). Left panel: Coomassie blue staining after SDS–PAGE of purified Bsu LigD. Right panel: autoradiography of corresponding protein-DNA adducts after the SDS–PAGE separation shown in left panel. When indicated, protein was previously incubated with 0.05 U of thrombin at 20°C for 60 min. ( B ) Adduct formation is dependent on the presence of an abasic site. Reactions were performed as in described in (A) but using as substrate 3.6 nM of the 3′ [α 32 P]3′-dAMP labeled oligonucleotide without removing the uracil ( absence of AP site ) or after treatment with E. coli UDG ( presence of AP site ), in the presence of either 100 mM NaBH 4 or NaCl (as indicated). Autoradiography of corresponding protein-DNA adduct after the SDS–PAGE separation is shown.

    Techniques Used: Labeling, Staining, SDS Page, Purification, Autoradiography, Incubation

    23) Product Images from "Phaeocystis globosa Virus DNA Polymerase X: a “Swiss Army knife”, Multifunctional DNA polymerase-lyase-ligase for Base Excision Repair"

    Article Title: Phaeocystis globosa Virus DNA Polymerase X: a “Swiss Army knife”, Multifunctional DNA polymerase-lyase-ligase for Base Excision Repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07378-3

    AP-lyase activity of PgVPolX . The depicted [ 32 P]3′-labeled uracil-containing oligonucleotide (top) was treated with E. coli UDG, leaving an intact AP site. The resulting AP-containing DNA (1 nM) was incubated in the presence of either E. coli EndoIII that incises 3′ to the AP site, or the indicated PgV-PolX for 10 min at 30 °C, as described in Materials and Methods. Position of products is indicated.
    Figure Legend Snippet: AP-lyase activity of PgVPolX . The depicted [ 32 P]3′-labeled uracil-containing oligonucleotide (top) was treated with E. coli UDG, leaving an intact AP site. The resulting AP-containing DNA (1 nM) was incubated in the presence of either E. coli EndoIII that incises 3′ to the AP site, or the indicated PgV-PolX for 10 min at 30 °C, as described in Materials and Methods. Position of products is indicated.

    Techniques Used: Activity Assay, Labeling, Incubation

    24) 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

    25) Product Images from "Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus"

    Article Title: Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2007.05.011

    DNA glycosylase/lyase activities of wild type and mutant MvNei1
    Figure Legend Snippet: DNA glycosylase/lyase activities of wild type and mutant MvNei1

    Techniques Used: Mutagenesis

    DNA glycosylase/lyase activities of mimiviral Nei proteins
    Figure Legend Snippet: DNA glycosylase/lyase activities of mimiviral Nei proteins

    Techniques Used:

    26) 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

    27) 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

    28) 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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr933

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

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

    30) Product Images from "Mutational Analysis of Arginine 276 in the Leucine-loop of Human Uracil-DNA Glycosylase *"

    Article Title: Mutational Analysis of Arginine 276 in the Leucine-loop of Human Uracil-DNA Glycosylase *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M407836200

    Ability of UNG and Arg 276 mutant proteins to form UV-catalyzed cross-links to [ 32 P]25-mer DNA A , samples (12 µl) containing 20 pmol of 5′-end 32 P-labeled oligonucleotide T-25-mer, U-25-mer, or dψU-25-mer without UNG ( lanes 1–3 , respectively) or with 40 pmol of UNG ( lanes 4–6 , respectively) were UV-irradiated for 30 min as described under “Experimental Procedures.” Following irradiation, samples were subjected to non-denaturing polyacrylamide gel electrophoresis; the gels were dried and analyzed using a PhosphorImager. The positions of the UNG × [ 32 P]DNA-25-mer cross-linked complex bands and free [ 32 P]DNA-25-mer bands are indicated by arrows . B , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min ( lanes 3–14 , respectively), reactions were analyzed as in A . Control reactions ( lanes 1 and 2 ), containing 20 pmol of [ 32 P]dψU-25-mer, were not irradiated. C , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer ( closed circles ), [ 32 P]T-25-mer ( closed squares ), or [ 32 P]U-25-mer ( closed triangles ), and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min, reactions were analyzed as in A , and the PhosphorImager data were quantified using the ImageQuant program. The cross-linking efficiency (%) was calculated by dividing the intensity of the UNG × [ 32 P]25-mer band by the sum of the [ 32 P]25-mer and UNG × [ 32 P]25-mer bands and multiplying by 100. D , reaction mixtures (12 µl) were prepared that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of each Arg 276 mutant enzyme. The reactions were UV-irradiated for 10 min and analyzed as described in C . The cross-linking efficiency of each mutant preparation, indicated by the corresponding single letter amino acid abbreviation, is compared with that of UNG. Error bars represent the standard deviation of three experiments.
    Figure Legend Snippet: Ability of UNG and Arg 276 mutant proteins to form UV-catalyzed cross-links to [ 32 P]25-mer DNA A , samples (12 µl) containing 20 pmol of 5′-end 32 P-labeled oligonucleotide T-25-mer, U-25-mer, or dψU-25-mer without UNG ( lanes 1–3 , respectively) or with 40 pmol of UNG ( lanes 4–6 , respectively) were UV-irradiated for 30 min as described under “Experimental Procedures.” Following irradiation, samples were subjected to non-denaturing polyacrylamide gel electrophoresis; the gels were dried and analyzed using a PhosphorImager. The positions of the UNG × [ 32 P]DNA-25-mer cross-linked complex bands and free [ 32 P]DNA-25-mer bands are indicated by arrows . B , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min ( lanes 3–14 , respectively), reactions were analyzed as in A . Control reactions ( lanes 1 and 2 ), containing 20 pmol of [ 32 P]dψU-25-mer, were not irradiated. C , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer ( closed circles ), [ 32 P]T-25-mer ( closed squares ), or [ 32 P]U-25-mer ( closed triangles ), and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min, reactions were analyzed as in A , and the PhosphorImager data were quantified using the ImageQuant program. The cross-linking efficiency (%) was calculated by dividing the intensity of the UNG × [ 32 P]25-mer band by the sum of the [ 32 P]25-mer and UNG × [ 32 P]25-mer bands and multiplying by 100. D , reaction mixtures (12 µl) were prepared that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of each Arg 276 mutant enzyme. The reactions were UV-irradiated for 10 min and analyzed as described in C . The cross-linking efficiency of each mutant preparation, indicated by the corresponding single letter amino acid abbreviation, is compared with that of UNG. Error bars represent the standard deviation of three experiments.

    Techniques Used: Mutagenesis, Labeling, Irradiation, Polyacrylamide Gel Electrophoresis, Standard Deviation

    Tertiary structure of human uracil-DNA glycosylase bound to DNA ). The DNA is shown in yellow and the view is looking into the major groove. A , three distinct amino acid sequences ( red tubes ) of the UNG* polypeptide backbone ( silver tubes ) are shown. The 4 Pro-Ser loop ( 165 PPPPS 169 ) and the Gly-Ser loop ( 246 GS 247 ). The Leu 272 loop ( 268 HPSPLSVYR 276 ), which contains Arg 276 ). Conserved amino acid residues (Gln 144 , Asn 204 , and His 268 ) in the UNG* binding pocket capture (“pull”) and stabilize the expelled extrahelical dψU. α-Helices are depicted as silver cylinders and β-sheets are illustrated as blue strands . B , ball-and-stick diagram of the UNG Leu 272 loop ( 271 PLSVYR 276 ) shown in silver and a portion of the oligonucleotide sequence 3′-CTA dψU-5′ shown in yellow . The ηN of the Arg 276 guanidinium side chain (nitrogen atoms, blue balls ) is shown as interacting ( black rippled lines ) with the 5′-phosphate of the cytosine residue (oxygen atom, red ball ), as stated for cleaved U·G DNA by Slupphaug et al. ). The εN participates in water-bridged (water, black ball ) hydrogen bonding ( dashed lines ) with the N3 of adenine (blue ball) and the carbonyl group ( red ball ) of Leu 272 as shown in Parikh et al. ). Structures were drawn with the Cn3D 4.0 software program using Protein Data Bank code 1EMH (MMDB 13471) deposited by Parikh et al. ) in the Molecular Modeling Data base of the National Center for Biotechnology Information.
    Figure Legend Snippet: Tertiary structure of human uracil-DNA glycosylase bound to DNA ). The DNA is shown in yellow and the view is looking into the major groove. A , three distinct amino acid sequences ( red tubes ) of the UNG* polypeptide backbone ( silver tubes ) are shown. The 4 Pro-Ser loop ( 165 PPPPS 169 ) and the Gly-Ser loop ( 246 GS 247 ). The Leu 272 loop ( 268 HPSPLSVYR 276 ), which contains Arg 276 ). Conserved amino acid residues (Gln 144 , Asn 204 , and His 268 ) in the UNG* binding pocket capture (“pull”) and stabilize the expelled extrahelical dψU. α-Helices are depicted as silver cylinders and β-sheets are illustrated as blue strands . B , ball-and-stick diagram of the UNG Leu 272 loop ( 271 PLSVYR 276 ) shown in silver and a portion of the oligonucleotide sequence 3′-CTA dψU-5′ shown in yellow . The ηN of the Arg 276 guanidinium side chain (nitrogen atoms, blue balls ) is shown as interacting ( black rippled lines ) with the 5′-phosphate of the cytosine residue (oxygen atom, red ball ), as stated for cleaved U·G DNA by Slupphaug et al. ). The εN participates in water-bridged (water, black ball ) hydrogen bonding ( dashed lines ) with the N3 of adenine (blue ball) and the carbonyl group ( red ball ) of Leu 272 as shown in Parikh et al. ). Structures were drawn with the Cn3D 4.0 software program using Protein Data Bank code 1EMH (MMDB 13471) deposited by Parikh et al. ) in the Molecular Modeling Data base of the National Center for Biotechnology Information.

    Techniques Used: Binding Assay, Sequencing, Software

    Specific uracil-DNA glycosylase activity of R276X mutant proteins ) and the Protein Assay reagent (Bio-Rad). The specific activity (units/mg) of UNG* and the R276X mutant enzymes was normalized to that of UNG (3.76 × 10 5 units/mg), which was defined as 100%. Arg 276 mutant proteins are denoted by single letter amino acid abbreviations. Error bars represent the standard deviation of four experimental determinations.
    Figure Legend Snippet: Specific uracil-DNA glycosylase activity of R276X mutant proteins ) and the Protein Assay reagent (Bio-Rad). The specific activity (units/mg) of UNG* and the R276X mutant enzymes was normalized to that of UNG (3.76 × 10 5 units/mg), which was defined as 100%. Arg 276 mutant proteins are denoted by single letter amino acid abbreviations. Error bars represent the standard deviation of four experimental determinations.

    Techniques Used: Activity Assay, Mutagenesis, Standard Deviation

    31) Product Images from "Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications"

    Article Title: Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.062082606

    Gene fragmentation. ( A ) Schematic of the CDH gene fragmentation process. PCR with TTP/dUTP mixtures is used to generate copies of the target gene in which uracil is randomly incorporated in place of thymine. The uracil-doped amplified DNA is subjected to a modified base-excision cascade in which uracil-DNA glycosylase excises the uracil bases generating abasic sites, which are cleaved by endonuclease IV, giving a single-strand nick that is converted to a double-strand break and blunt-ended by S1 nuclease. As the reaction cascade is initiated only at uracils, whose distribution along the sequence and among the PCR reaction products is random, the cascade generates a random and unbiased library of gene fragments, whose size distribution is solely dictated by the TTP/dUTP ratio. ( B ) dUTP-dose dependent fragmentation. SYBR-Safe stained 1% agarose gel of an ∼2.2-kb human p85α PCR-amplified cDNA ( right- hand lane), alongside the products of CDH fragmentation reactions using increasing amounts of dUTP (as percent of total TTP+dUTP concentration). The progressive decrease in modal size of the DNA distribution with increasing dUTP concentration is clearly seen.
    Figure Legend Snippet: Gene fragmentation. ( A ) Schematic of the CDH gene fragmentation process. PCR with TTP/dUTP mixtures is used to generate copies of the target gene in which uracil is randomly incorporated in place of thymine. The uracil-doped amplified DNA is subjected to a modified base-excision cascade in which uracil-DNA glycosylase excises the uracil bases generating abasic sites, which are cleaved by endonuclease IV, giving a single-strand nick that is converted to a double-strand break and blunt-ended by S1 nuclease. As the reaction cascade is initiated only at uracils, whose distribution along the sequence and among the PCR reaction products is random, the cascade generates a random and unbiased library of gene fragments, whose size distribution is solely dictated by the TTP/dUTP ratio. ( B ) dUTP-dose dependent fragmentation. SYBR-Safe stained 1% agarose gel of an ∼2.2-kb human p85α PCR-amplified cDNA ( right- hand lane), alongside the products of CDH fragmentation reactions using increasing amounts of dUTP (as percent of total TTP+dUTP concentration). The progressive decrease in modal size of the DNA distribution with increasing dUTP concentration is clearly seen.

    Techniques Used: Polymerase Chain Reaction, Amplification, Modification, Sequencing, Staining, Agarose Gel Electrophoresis, Concentration Assay

    32) Product Images from "A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine"

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq982

    An unusual sequence insertion is present in the DNA glycosylase domain of members of the DML family. ( A ) Schematic diagram showing ROS1 regions conserved among DML proteins. ( B ) Multiple sequence alignment of DML proteins and several HhH-GPD superfamily members. Listed above the primary sequence are indicated secondary structure assignments from the ROS1 model prediction shown in (C), colored according to regions shown in (A). The helix–hairpin–helix of the HhH-GPD motif is shown in cyan. ROS1 amino acids mutated in this study are indicated by inverted triangles and highlighted in green (Q584 and W1012), blue (F589 and Y1028), yellow (T606 and D611) or red (Q607 and N608). The lysine residue that is diagnostic of bifunctional glycosylase/lyase activity, and the conserved aspartic acid residue in the active site are indicated by asterisks. The HhH-GPD and the [4Fe–4S] cluster loop (FCL) motifs are boxed. Names of organisms are abbreviated as follows: Ath, Arabidopsis thaliana ; Nta, Nicotiana tabacum ; Bst, Bacillus stearothermophilus ; Eco, Escherichia coli ; Mth, Methanobacterium thermoautotrophicum ; Mmu, Mus musculus ; Hsa, Homo sapiens . Genbank accession numbers are: Ath ROS1: AAP37178; Ath DME: ABC61677; Nta ROS1: BAF52855; Bst EndoIII: 1P59; Eco EndoIII: P20625; Mth Mig: NP_039762; Eco MutY: NP_417436; Mmu MBD4: 1NGN; Hsa OGG1: O15527; Eco AlkA: P04395. ( C ) Ribbon diagrams of the structural model for the DNA glycosylase domain of ROS1 and the crystallographic Bst EndoIII structure used as template. Structural elements are colored as in (A). The duplex DNA is shown in orange. Nucleic acid coordinates extracted from the Bst EndoIII-DNA trapped complex were used to superimpose a DNA structure with a flipped-out AP site analog onto the ROS1 model. ( D ) Close-up view of the ROS1 model. Mutated residues are shown as sticks and colored according to (B). The conserved lysine and aspartic acid residues are shown in magenta.
    Figure Legend Snippet: An unusual sequence insertion is present in the DNA glycosylase domain of members of the DML family. ( A ) Schematic diagram showing ROS1 regions conserved among DML proteins. ( B ) Multiple sequence alignment of DML proteins and several HhH-GPD superfamily members. Listed above the primary sequence are indicated secondary structure assignments from the ROS1 model prediction shown in (C), colored according to regions shown in (A). The helix–hairpin–helix of the HhH-GPD motif is shown in cyan. ROS1 amino acids mutated in this study are indicated by inverted triangles and highlighted in green (Q584 and W1012), blue (F589 and Y1028), yellow (T606 and D611) or red (Q607 and N608). The lysine residue that is diagnostic of bifunctional glycosylase/lyase activity, and the conserved aspartic acid residue in the active site are indicated by asterisks. The HhH-GPD and the [4Fe–4S] cluster loop (FCL) motifs are boxed. Names of organisms are abbreviated as follows: Ath, Arabidopsis thaliana ; Nta, Nicotiana tabacum ; Bst, Bacillus stearothermophilus ; Eco, Escherichia coli ; Mth, Methanobacterium thermoautotrophicum ; Mmu, Mus musculus ; Hsa, Homo sapiens . Genbank accession numbers are: Ath ROS1: AAP37178; Ath DME: ABC61677; Nta ROS1: BAF52855; Bst EndoIII: 1P59; Eco EndoIII: P20625; Mth Mig: NP_039762; Eco MutY: NP_417436; Mmu MBD4: 1NGN; Hsa OGG1: O15527; Eco AlkA: P04395. ( C ) Ribbon diagrams of the structural model for the DNA glycosylase domain of ROS1 and the crystallographic Bst EndoIII structure used as template. Structural elements are colored as in (A). The duplex DNA is shown in orange. Nucleic acid coordinates extracted from the Bst EndoIII-DNA trapped complex were used to superimpose a DNA structure with a flipped-out AP site analog onto the ROS1 model. ( D ) Close-up view of the ROS1 model. Mutated residues are shown as sticks and colored according to (B). The conserved lysine and aspartic acid residues are shown in magenta.

    Techniques Used: Sequencing, Diagnostic Assay, Activity Assay

    T606 and D611 are essential for ROS1 DNA glycosylase activity. ( A ) The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. Samples were treated with or without NaOH 100 mM, and immediately transferred to 90°C for 10 min. Products were separated in a 12% denaturing polyacrylamide gel and the amounts of incised oligonucleotide were quantified by fluorescent scanning. ( B ) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair, either in the absence or the presence of human APE I (5 U), as indicated. Products were separated in a 12% denaturing polyacrylamide gel and the incised products were detected by fluorescent scanning. ( C ) A double-stranded oligonucleotide substrate containing an AP site opposite G (200 nM) was incubated at 30°C either in the absence of enzyme or in the presence of purified WT ROS1, T606L or D611V (100 nM). Reactions were stopped at the indicated times, products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are means ± SE (error bars) from two independent experiments. The asterisks indicate that the incision levels were significantly different ( P
    Figure Legend Snippet: T606 and D611 are essential for ROS1 DNA glycosylase activity. ( A ) The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. Samples were treated with or without NaOH 100 mM, and immediately transferred to 90°C for 10 min. Products were separated in a 12% denaturing polyacrylamide gel and the amounts of incised oligonucleotide were quantified by fluorescent scanning. ( B ) Purified WT ROS1 or mutant variants (20 nM) were incubated at 30°C for 2 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair, either in the absence or the presence of human APE I (5 U), as indicated. Products were separated in a 12% denaturing polyacrylamide gel and the incised products were detected by fluorescent scanning. ( C ) A double-stranded oligonucleotide substrate containing an AP site opposite G (200 nM) was incubated at 30°C either in the absence of enzyme or in the presence of purified WT ROS1, T606L or D611V (100 nM). Reactions were stopped at the indicated times, products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are means ± SE (error bars) from two independent experiments. The asterisks indicate that the incision levels were significantly different ( P

    Techniques Used: Activity Assay, Purification, Mutagenesis, Incubation

    33) 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

    34) Product Images from "Efficient processing of abasic sites by bacterial nonhomologous end-joining Ku proteins"

    Article Title: Efficient processing of abasic sites by bacterial nonhomologous end-joining Ku proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1029

    AP-lyase activity is also present in the Ku protein from the Gram- bacteria P. aeruginosa . ( A ) Formation of Pae Ku–DNA adducts. Top panels , expression of the recombinant Pae Ku. For a better visualization of the induced Pae Ku, 15 and 6 μg of total protein from the cellular extracts were loaded in the noninduced (NI) and in the induced (Ind.) lanes, respectively. Bottom panels , autoradiography of corresponding protein–DNA adducts after the SDS-PAGE separation. Reactions were performed as described in Materials and Methods, incubating either 1.7 μg of the noninduced bacterial extract or 0.7 μg of the induced extract with 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide containing an AP site at position 16 (after treatment with Escherichia coli UDG), in the presence of 100 mM NaBH 4 . When indicated, the bacterial extracts were previously incubated with 0.05 U of thrombin at 20°C for 60 min. ( B ) AP-lyase activity of purified Pae Ku. 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide containing an AP site at position 16 (after treatment with E. coli UDG) was incubated in the presence of either Bsu Ku (228 nM), or increasing concentrations (14, 28, 57, 114 and 228 nM) of Pae Ku for 1 h at 30°C, as described in Materials and Methods. c : control DNA after treatment with E. coli UDG. After incubation, samples were analysed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated.
    Figure Legend Snippet: AP-lyase activity is also present in the Ku protein from the Gram- bacteria P. aeruginosa . ( A ) Formation of Pae Ku–DNA adducts. Top panels , expression of the recombinant Pae Ku. For a better visualization of the induced Pae Ku, 15 and 6 μg of total protein from the cellular extracts were loaded in the noninduced (NI) and in the induced (Ind.) lanes, respectively. Bottom panels , autoradiography of corresponding protein–DNA adducts after the SDS-PAGE separation. Reactions were performed as described in Materials and Methods, incubating either 1.7 μg of the noninduced bacterial extract or 0.7 μg of the induced extract with 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide containing an AP site at position 16 (after treatment with Escherichia coli UDG), in the presence of 100 mM NaBH 4 . When indicated, the bacterial extracts were previously incubated with 0.05 U of thrombin at 20°C for 60 min. ( B ) AP-lyase activity of purified Pae Ku. 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide containing an AP site at position 16 (after treatment with E. coli UDG) was incubated in the presence of either Bsu Ku (228 nM), or increasing concentrations (14, 28, 57, 114 and 228 nM) of Pae Ku for 1 h at 30°C, as described in Materials and Methods. c : control DNA after treatment with E. coli UDG. After incubation, samples were analysed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated.

    Techniques Used: Activity Assay, Expressing, Recombinant, Autoradiography, SDS Page, Labeling, Incubation, Purification, Polyacrylamide Gel Electrophoresis

    Bsu Ku is endowed with AP-lyase activity. ( A ) The [ 32 P]5′-labeled uracil-containing oligonucleotide was treated with Escherichia coli UDG, leaving an intact AP site. The resulting AP-containing DNA was incubated in the presence of either h APE1 that cleaves 5′ to the AP site, EndoIII that incises 3′ to the AP site or increasing concentrations (9, 18, 36, 72 and 142 nM) of Bsu Ku for 1 h at 30°C, as described in Materials and Methods. c : control DNA after treatment with E. coli UDG. After incubation samples were analysed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. ( B ) The 3′[ 32 P]3′-dAMP labeled oligonucleotide containing the lyase-resistant analog tetrahydrofuran (THF) at position 19 was incubated in the presence of either h APE1, EndoIII or Bsu Ku (228 nM) as described above. c : control DNA incubated in the absence of proteins. Position corresponding to product (16mer 5′-dRP) is indicated.
    Figure Legend Snippet: Bsu Ku is endowed with AP-lyase activity. ( A ) The [ 32 P]5′-labeled uracil-containing oligonucleotide was treated with Escherichia coli UDG, leaving an intact AP site. The resulting AP-containing DNA was incubated in the presence of either h APE1 that cleaves 5′ to the AP site, EndoIII that incises 3′ to the AP site or increasing concentrations (9, 18, 36, 72 and 142 nM) of Bsu Ku for 1 h at 30°C, as described in Materials and Methods. c : control DNA after treatment with E. coli UDG. After incubation samples were analysed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. ( B ) The 3′[ 32 P]3′-dAMP labeled oligonucleotide containing the lyase-resistant analog tetrahydrofuran (THF) at position 19 was incubated in the presence of either h APE1, EndoIII or Bsu Ku (228 nM) as described above. c : control DNA incubated in the absence of proteins. Position corresponding to product (16mer 5′-dRP) is indicated.

    Techniques Used: Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    Formation of Bsu Ku-DNA adducts. ( A ) Dependence of Bsu Ku-DNA cross-link on NaBH 4 . Reactions were performed as described in Materials and Methods, incubating 228 nM Bsu Ku with 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide containing an AP site at position 16 (after treatment with Escherichia coli UDG), in the presence of either 100 mM NaBH 4 or NaCl (as indicated). Top panels , Coomassie blue staining after SDS-PAGE of purified Bsu Ku. Bottom panels , autoradiography of corresponding protein–DNA adducts after the SDS-PAGE separation shown in top panels. When indicated, protein was previously incubated with 0.05 U of thrombin at 20°C for 60 min. ( B ) Adduct formation is dependent on the presence of an abasic site. Reactions were performed as described in (A) but using as substrate 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide without removing the uracil at position 16 (absence of AP site) or after treatment with E. coli UDG (presence of AP site), in the presence of either 100 mM NaBH 4 or NaCl (as indicated). Autoradiography of corresponding protein–DNA adduct after the SDS-PAGE separation is shown.
    Figure Legend Snippet: Formation of Bsu Ku-DNA adducts. ( A ) Dependence of Bsu Ku-DNA cross-link on NaBH 4 . Reactions were performed as described in Materials and Methods, incubating 228 nM Bsu Ku with 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide containing an AP site at position 16 (after treatment with Escherichia coli UDG), in the presence of either 100 mM NaBH 4 or NaCl (as indicated). Top panels , Coomassie blue staining after SDS-PAGE of purified Bsu Ku. Bottom panels , autoradiography of corresponding protein–DNA adducts after the SDS-PAGE separation shown in top panels. When indicated, protein was previously incubated with 0.05 U of thrombin at 20°C for 60 min. ( B ) Adduct formation is dependent on the presence of an abasic site. Reactions were performed as described in (A) but using as substrate 3.1 nM of the 3′[ 32 P]3′-dAMP labeled 35mer oligonucleotide without removing the uracil at position 16 (absence of AP site) or after treatment with E. coli UDG (presence of AP site), in the presence of either 100 mM NaBH 4 or NaCl (as indicated). Autoradiography of corresponding protein–DNA adduct after the SDS-PAGE separation is shown.

    Techniques Used: Labeling, Staining, SDS Page, Purification, Autoradiography, Incubation

    Bsu Ku AP-lyase on protruding ends. The assay was performed as described in Materials and Methods. The substrates containing a uracil at the specified position within the 5′- (upper panels) or 3′- (lower panels) protruding ends were incubated with Escherichia coli UDG ( c ) to create an AP site in nearly all DNA molecules. After incubation of the AP-containing molecules with 2 units of E. coli Endo III (Endo), or 142 nM Bsu Ku for 30 min at 30°C, samples were analysed by 8 M urea-20% PAGE and autoradiography, as described in Materials and Methods. Positions corresponding to the products are indicated. Asterisks indicate either the 32 P-5′ or the [ 32 P]3′dAMP-3′end.
    Figure Legend Snippet: Bsu Ku AP-lyase on protruding ends. The assay was performed as described in Materials and Methods. The substrates containing a uracil at the specified position within the 5′- (upper panels) or 3′- (lower panels) protruding ends were incubated with Escherichia coli UDG ( c ) to create an AP site in nearly all DNA molecules. After incubation of the AP-containing molecules with 2 units of E. coli Endo III (Endo), or 142 nM Bsu Ku for 30 min at 30°C, samples were analysed by 8 M urea-20% PAGE and autoradiography, as described in Materials and Methods. Positions corresponding to the products are indicated. Asterisks indicate either the 32 P-5′ or the [ 32 P]3′dAMP-3′end.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    35) Product Images from "Unencumbered Pol β lyase activity in nucleosome core particles"

    Article Title: Unencumbered Pol β lyase activity in nucleosome core particles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx593

    Pol β dRP lyase activity assay. ( A ) DNA oligo (15-mer) was 5′-end labeled with γ- 32 P-ATP. Experiments were conducted as described in Materials and Methods. The reaction with Pol β was initiated ∼30 s after UDG incubation. A negative control was included to subtract the spontaneous loss of the 5′-dRP. A heat control (H), as described in Materials and Methods, was included to determine the actual dRP substrate generated after the 5.5-min UDG incubation. Full length Pol β (FL) or the N-terminal domain of Pol β (8 kDa) were used to determine dRP lyase activity. A representative phosphor image of a 20% polyacrylamide denaturing gel of the replicates plotted in panel (B) is shown. ( B ) NCP crystal structure pdb file 3LZ0 ( 37 ) was modified to show the structural location of the 5′-dRP group (black spheres). This is the back view of that shown in Figure 1 . Data represent the mean of three independent experiments ± SD. Kinetic parameters are provided in Table 2 (full length Pol β) and Table 3 (8 kDa domain of Pol β). As shown in Table 2 , the ratio (DNA/NCP) of the enzymatic rates for this 5′-end labeled substrate is comparable to the ratio found by the 3′-end labeling assay ( Supplementary Figure S3 ). The rate of spontaneous loss, estimated from the –Pol β control, for the NCP is ∼3.2 nM/min, which is 8-fold faster than the rate in DNA of 0.4 nM/min.
    Figure Legend Snippet: Pol β dRP lyase activity assay. ( A ) DNA oligo (15-mer) was 5′-end labeled with γ- 32 P-ATP. Experiments were conducted as described in Materials and Methods. The reaction with Pol β was initiated ∼30 s after UDG incubation. A negative control was included to subtract the spontaneous loss of the 5′-dRP. A heat control (H), as described in Materials and Methods, was included to determine the actual dRP substrate generated after the 5.5-min UDG incubation. Full length Pol β (FL) or the N-terminal domain of Pol β (8 kDa) were used to determine dRP lyase activity. A representative phosphor image of a 20% polyacrylamide denaturing gel of the replicates plotted in panel (B) is shown. ( B ) NCP crystal structure pdb file 3LZ0 ( 37 ) was modified to show the structural location of the 5′-dRP group (black spheres). This is the back view of that shown in Figure 1 . Data represent the mean of three independent experiments ± SD. Kinetic parameters are provided in Table 2 (full length Pol β) and Table 3 (8 kDa domain of Pol β). As shown in Table 2 , the ratio (DNA/NCP) of the enzymatic rates for this 5′-end labeled substrate is comparable to the ratio found by the 3′-end labeling assay ( Supplementary Figure S3 ). The rate of spontaneous loss, estimated from the –Pol β control, for the NCP is ∼3.2 nM/min, which is 8-fold faster than the rate in DNA of 0.4 nM/min.

    Techniques Used: Activity Assay, Labeling, Incubation, Negative Control, Generated, Modification, End Labeling

    36) 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

    37) Product Images from "Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations"

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    Journal: eLife

    doi: 10.7554/eLife.51605

    Purification and activity of NEIL2. ( A ) Purified NEIL2-His 6 (50 ng) from E. coli was subjected to NuPAGE and stained with Coomassie blue. ( B ) Activity of purified NEIL2-His 6 on 5’-[ 32 P]-labeled oligonucleotides (51 nt) containing: hydroxyuracil (OHU), OHU/G, U, or U/G. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. S, substrate; P, product. ( C ) Validation of UDG-generated AP sites from 5’-[ 32 P]-labeled single-stranded and double-stranded oligonucleotides (35 nt) by treatment with NaOH. AP sites are lysed by alkali treatment. ( D ) NEIL2 cleaves Fluorescein (Fluor)-labeled U-containing single strand oligonucleotide (39 nt) in the presence of UDG. S, substrate; P, product.
    Figure Legend Snippet: Purification and activity of NEIL2. ( A ) Purified NEIL2-His 6 (50 ng) from E. coli was subjected to NuPAGE and stained with Coomassie blue. ( B ) Activity of purified NEIL2-His 6 on 5’-[ 32 P]-labeled oligonucleotides (51 nt) containing: hydroxyuracil (OHU), OHU/G, U, or U/G. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. S, substrate; P, product. ( C ) Validation of UDG-generated AP sites from 5’-[ 32 P]-labeled single-stranded and double-stranded oligonucleotides (35 nt) by treatment with NaOH. AP sites are lysed by alkali treatment. ( D ) NEIL2 cleaves Fluorescein (Fluor)-labeled U-containing single strand oligonucleotide (39 nt) in the presence of UDG. S, substrate; P, product.

    Techniques Used: Purification, Activity Assay, Staining, Labeling, Generated

    Reaction mechanisms for the APE1 and NEIL2-PNKP pathways. Glycosylase UDG removes U and generates an AP site on U/G mismatch (only top strand is shown). For the APE1 pathway, APE1 hydrolyzes the deoxyribose-phosphate backbone and generates a 5’-deoxyribophosphate (5’dRP) and a 3’-hydroxyl (3’OH). DNA Polymerase β (Polβ) adds a C to the 3’OH and removes the 5’dRP. For the NEIL2-PNKP pathway, NEIL2 catalyzes β,δ-elimination step by step at the AP site and generates a 5’-phosphate (5’P) and a 3’P. PNKP phosphatase hydrolyzes 3’P to generate a 3’OH primer for Polβ.
    Figure Legend Snippet: Reaction mechanisms for the APE1 and NEIL2-PNKP pathways. Glycosylase UDG removes U and generates an AP site on U/G mismatch (only top strand is shown). For the APE1 pathway, APE1 hydrolyzes the deoxyribose-phosphate backbone and generates a 5’-deoxyribophosphate (5’dRP) and a 3’-hydroxyl (3’OH). DNA Polymerase β (Polβ) adds a C to the 3’OH and removes the 5’dRP. For the NEIL2-PNKP pathway, NEIL2 catalyzes β,δ-elimination step by step at the AP site and generates a 5’-phosphate (5’P) and a 3’P. PNKP phosphatase hydrolyzes 3’P to generate a 3’OH primer for Polβ.

    Techniques Used:

    38) Product Images from "MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes"

    Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

    Journal: bioRxiv

    doi: 10.1101/2020.02.13.947598

    Extent of leaf variegation observed for different msh1 mutant alleles. A. An example of an msh1 mutant (CS3372) individual with a leaf-variegation phenotype. B. Values represent the percent of individuals in an F3 family from a homozygous mutant F2 parent that showed visible leaf variegation at time of harvest for mitochondrial and plastid DNA extraction. Means and standard errors are from three replicate F3 families from each mutant line (see Fig. 2 ). Between 45 and 66 individuals were scored for each family. Lowercase letters indicate significant differences between alleles based on a Tukey’s HSD test. Consistent with its lower rate of observed sequence and structural variation in cytoplasmic genomes, the SALK_046763 msh1 mutant line exhibited less severe phenotypic effects.
    Figure Legend Snippet: Extent of leaf variegation observed for different msh1 mutant alleles. A. An example of an msh1 mutant (CS3372) individual with a leaf-variegation phenotype. B. Values represent the percent of individuals in an F3 family from a homozygous mutant F2 parent that showed visible leaf variegation at time of harvest for mitochondrial and plastid DNA extraction. Means and standard errors are from three replicate F3 families from each mutant line (see Fig. 2 ). Between 45 and 66 individuals were scored for each family. Lowercase letters indicate significant differences between alleles based on a Tukey’s HSD test. Consistent with its lower rate of observed sequence and structural variation in cytoplasmic genomes, the SALK_046763 msh1 mutant line exhibited less severe phenotypic effects.

    Techniques Used: Mutagenesis, DNA Extraction, Sequencing

    Crossing design to test candidate nuclear genes involved in RRR of cytoplasmic genomes. Using a wild-type maternal plant (black) and either a homozygous mutant (red) or heterozygous pollen donor, we generated a heterozygous F1 individual that carried cytoplasmic genomes inherited from a wild-type lineage (as indicated by the black mitochondrion). After selfing the F1, we genotyped the resulting F2 progeny to identify three homozygous mutants and three homozygous wild-type individuals. Given that the mutations in candidate RRR genes are expected to be recessive, the F2 generation would be the first in which the sampled cytoplasmic genomes were exposed to the effects (red asterisks) of these mutants. The identified F2 individuals were each allowed to self-fertilize and set seed to produce multiple F3 families that all inherited their cytoplasmic genomes from the same F1 grandparent. The F3 families were used for purification of mitochondrial and plastid DNA for duplex sequencing. Sequencing was performed on three replicate families for each genotype. Arabidopsis silhouette image is from PhyloPic (Mason McNair).
    Figure Legend Snippet: Crossing design to test candidate nuclear genes involved in RRR of cytoplasmic genomes. Using a wild-type maternal plant (black) and either a homozygous mutant (red) or heterozygous pollen donor, we generated a heterozygous F1 individual that carried cytoplasmic genomes inherited from a wild-type lineage (as indicated by the black mitochondrion). After selfing the F1, we genotyped the resulting F2 progeny to identify three homozygous mutants and three homozygous wild-type individuals. Given that the mutations in candidate RRR genes are expected to be recessive, the F2 generation would be the first in which the sampled cytoplasmic genomes were exposed to the effects (red asterisks) of these mutants. The identified F2 individuals were each allowed to self-fertilize and set seed to produce multiple F3 families that all inherited their cytoplasmic genomes from the same F1 grandparent. The F3 families were used for purification of mitochondrial and plastid DNA for duplex sequencing. Sequencing was performed on three replicate families for each genotype. Arabidopsis silhouette image is from PhyloPic (Mason McNair).

    Techniques Used: Mutagenesis, Generated, Purification, Sequencing

    Intact MSH1 transcripts but reduced expression level in homozygous SALK_046763 msh1 mutants. A. Sanger trace from cDNA sequencing confirms that properly spliced transcripts are present in SALK_046763 msh1 mutants despite the large T-DNA insertion in intron 8. The vertical line below the trace indicates the location of the expected splice junction between exons 8 and 9. B. ΔC T values are calculated based on the difference in quantitative reverse-transcriptase PCR (qRT-PCR) threshold cycle value for each indicated MSH1 marker and the geometric mean of the threshold cycle values from two reference genes ( UBC and UBC9 ). Means and standard errors are from four biological replicates (F4 plants derived from crossing design described in Fig. 2 ), each of which is based on the mean of three technical replicates. The SALK_046763 mutants exhibit higher ΔC T (indicating lower MSH1 expression). Both MSH1 markers indicate a similar shift in ΔC T values (2.3 cycles for exons 8/9 and 2.5 cycles for exon 16), corresponding to an approximately 5-fold difference in transcript abundance. Significant differences between mutant and wild type genotypes at a level of P
    Figure Legend Snippet: Intact MSH1 transcripts but reduced expression level in homozygous SALK_046763 msh1 mutants. A. Sanger trace from cDNA sequencing confirms that properly spliced transcripts are present in SALK_046763 msh1 mutants despite the large T-DNA insertion in intron 8. The vertical line below the trace indicates the location of the expected splice junction between exons 8 and 9. B. ΔC T values are calculated based on the difference in quantitative reverse-transcriptase PCR (qRT-PCR) threshold cycle value for each indicated MSH1 marker and the geometric mean of the threshold cycle values from two reference genes ( UBC and UBC9 ). Means and standard errors are from four biological replicates (F4 plants derived from crossing design described in Fig. 2 ), each of which is based on the mean of three technical replicates. The SALK_046763 mutants exhibit higher ΔC T (indicating lower MSH1 expression). Both MSH1 markers indicate a similar shift in ΔC T values (2.3 cycles for exons 8/9 and 2.5 cycles for exon 16), corresponding to an approximately 5-fold difference in transcript abundance. Significant differences between mutant and wild type genotypes at a level of P

    Techniques Used: Expressing, Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Derivative Assay, Mutagenesis

    39) Product Images from "Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification"

    Article Title: Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification

    Journal: ACS Omega

    doi: 10.1021/acsomega.8b03376

    (a) Real-time fluorescence detection UDG under different conditions: (1) negative control test only containing TP, cPLP, and dNTPs (including dUTP) without UDG and Endo IV in RCA reaction, (2) positive test with UDG and Endo IV in RCA reaction, (3) negative control test with UDG but no Endo IV in RCA reaction, (4) negative control test with Endo IV but no UDG in RCA reaction, and (5) negative control test without dUTP in RCA reaction. (b). Agarose gel electrophoresis results of RCA products, lanes 1–5 correspond to curves 1–5 in (a), respectively. The concentrations of reagents were 0.01 U UDG, 1 nM TP, 10 nM cPLP, 10 U Endo IV, 10 U phi29 DNA polymerase, 1 mM dNTP (with or without 1:9 dTTP/dUTP ).
    Figure Legend Snippet: (a) Real-time fluorescence detection UDG under different conditions: (1) negative control test only containing TP, cPLP, and dNTPs (including dUTP) without UDG and Endo IV in RCA reaction, (2) positive test with UDG and Endo IV in RCA reaction, (3) negative control test with UDG but no Endo IV in RCA reaction, (4) negative control test with Endo IV but no UDG in RCA reaction, and (5) negative control test without dUTP in RCA reaction. (b). Agarose gel electrophoresis results of RCA products, lanes 1–5 correspond to curves 1–5 in (a), respectively. The concentrations of reagents were 0.01 U UDG, 1 nM TP, 10 nM cPLP, 10 U Endo IV, 10 U phi29 DNA polymerase, 1 mM dNTP (with or without 1:9 dTTP/dUTP ).

    Techniques Used: Fluorescence, Negative Control, Agarose Gel Electrophoresis

    40) 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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.0030135

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

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

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm1053

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

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

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

    ( A ) Sequence design for the investigation of uracil and abasic site cleavage on microarrays. Each sequence consists of a dT 15 -linker, then a 30mer with either no dUs (control) or an increasing number of dU-incorporations (from 1 to 9) replacing dTs in the following sequence: 5′-TTA CCA TAG AAT CAT GTG CCA TAC ATC ATC-3′. At the 5′-end, a control 25mer is synthesized (QC25), serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). The cleavage process was monitored by recording the hybridization-based fluorescence intensity before and after the UDG-mediated cleavage of uracil nucleotides. ( B ) Small excerpt (ca. 7% of the total synthesis area) of fluorescence scans before and after enzyme exposure. The scans show the fluorescence intensity, resulting from hybridization to a labelled, complementary oligonucleotide. The microarrays were scanned at 5 µm resolution. ( C ) Decrease in fluorescence intensity for the UDG-mediated uracil excision (thus generating abasic sites) as a function of the number of dU nucleotide incorporations per DNA substrate. The actual cleavage efficiencies correlate with the loss of fluorescence intensity resulting from DNA substrate cleavage. The array was incubated for one hour with UDG and the generated abasic sites were subsequently cleaved under alkaline conditions. The decrease in fluorescence intensity was recorded and normalized to the control strand (U0). The normalized intensities, indicated in arbitrary units, were plotted over the number of dUs per DNA substrate. Error bars are SD.

    Journal: Scientific Reports

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries

    doi: 10.1038/s41598-019-54044-x

    Figure Lengend Snippet: ( A ) Sequence design for the investigation of uracil and abasic site cleavage on microarrays. Each sequence consists of a dT 15 -linker, then a 30mer with either no dUs (control) or an increasing number of dU-incorporations (from 1 to 9) replacing dTs in the following sequence: 5′-TTA CCA TAG AAT CAT GTG CCA TAC ATC ATC-3′. At the 5′-end, a control 25mer is synthesized (QC25), serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). The cleavage process was monitored by recording the hybridization-based fluorescence intensity before and after the UDG-mediated cleavage of uracil nucleotides. ( B ) Small excerpt (ca. 7% of the total synthesis area) of fluorescence scans before and after enzyme exposure. The scans show the fluorescence intensity, resulting from hybridization to a labelled, complementary oligonucleotide. The microarrays were scanned at 5 µm resolution. ( C ) Decrease in fluorescence intensity for the UDG-mediated uracil excision (thus generating abasic sites) as a function of the number of dU nucleotide incorporations per DNA substrate. The actual cleavage efficiencies correlate with the loss of fluorescence intensity resulting from DNA substrate cleavage. The array was incubated for one hour with UDG and the generated abasic sites were subsequently cleaved under alkaline conditions. The decrease in fluorescence intensity was recorded and normalized to the control strand (U0). The normalized intensities, indicated in arbitrary units, were plotted over the number of dUs per DNA substrate. Error bars are SD.

    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).

    Techniques: Sequencing, Synthesized, Hybridization, Fluorescence, Incubation, Generated

    Schematic illustration of the sequence design for the investigation of E. coli UDG sequence dependences on single- ( A ) and double-stranded DNA substrates. ( B ) In order to investigate the UDG sequence dependence, a single dU is incorporated into a DNA strand and enclosed by 3 permuted bases on each side. A The design for the study of UDG sequence dependence on single-stranded DNA substrates consists of a 15mer dT-linker, a single dU enclosed by 3 permuted bases on each side, followed by a 5′ 25mer sequence (QC25) serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). B For the study of UDG sequence dependence on double-stranded DNA substrates, the sequences were designed to form a hairpin loop. The resulting strands consisted of a 15mer dT-linker, a 11-nt stem, equivalent to the single-stranded design, containing the variable region flanked by dG·dC base pairs, a 4-nt loop followed by the complementary 11nt strand. At the 5′ end, a hybridizable 25mer target sequence (QC25) was synthesized.

    Journal: Scientific Reports

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries

    doi: 10.1038/s41598-019-54044-x

    Figure Lengend Snippet: Schematic illustration of the sequence design for the investigation of E. coli UDG sequence dependences on single- ( A ) and double-stranded DNA substrates. ( B ) In order to investigate the UDG sequence dependence, a single dU is incorporated into a DNA strand and enclosed by 3 permuted bases on each side. A The design for the study of UDG sequence dependence on single-stranded DNA substrates consists of a 15mer dT-linker, a single dU enclosed by 3 permuted bases on each side, followed by a 5′ 25mer sequence (QC25) serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). B For the study of UDG sequence dependence on double-stranded DNA substrates, the sequences were designed to form a hairpin loop. The resulting strands consisted of a 15mer dT-linker, a 11-nt stem, equivalent to the single-stranded design, containing the variable region flanked by dG·dC base pairs, a 4-nt loop followed by the complementary 11nt strand. At the 5′ end, a hybridizable 25mer target sequence (QC25) was synthesized.

    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).

    Techniques: Sequencing, Hybridization, Synthesized

    (Left) Schematic illustration of the UDG-mediated generation of single nucleotide gaps on nucleic acid strands. In a first step, UDG catalyzes the excision of uracil, leading to the formation of an abasic site. This AP-site can then either be cleaved by the lyase activity of specific endonucleases, or chemically. The USER enzyme, a mixture of UDG and Endonuclease VIII, combines AP-site formation and cleavage in a single solution. (Right) Molecular structures indicating the generation of a single nucleotide gap/strand cleavage via a β- and a subsequent δ-elimination reaction. First, UDG hydrolyzes the glycosidic bond from the uracil-containing DNA strand. The ribose at the apyrimidinic site lacks a glycosidic bond and is therefore highly unstable and converts rapidly into its reactive open-chain aldehyde, its hemiacetal or its hydrate form. The lyase activity of AP-endonucleases, or the exposure to either basic or acidic conditions, initiates a β-elimination reaction, resulting in the cleavage of the phosphodiester backbone 3′ to the AP-site and the formation of an α,β-unsaturated aldehyde. Subsequent δ-elimination induces DNA strand cleavage 5′ to the AP-site resulting in the generation of a single-nucleotide gap in dsDNA or strand cleavage in ssDNA.

    Journal: Scientific Reports

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries

    doi: 10.1038/s41598-019-54044-x

    Figure Lengend Snippet: (Left) Schematic illustration of the UDG-mediated generation of single nucleotide gaps on nucleic acid strands. In a first step, UDG catalyzes the excision of uracil, leading to the formation of an abasic site. This AP-site can then either be cleaved by the lyase activity of specific endonucleases, or chemically. The USER enzyme, a mixture of UDG and Endonuclease VIII, combines AP-site formation and cleavage in a single solution. (Right) Molecular structures indicating the generation of a single nucleotide gap/strand cleavage via a β- and a subsequent δ-elimination reaction. First, UDG hydrolyzes the glycosidic bond from the uracil-containing DNA strand. The ribose at the apyrimidinic site lacks a glycosidic bond and is therefore highly unstable and converts rapidly into its reactive open-chain aldehyde, its hemiacetal or its hydrate form. The lyase activity of AP-endonucleases, or the exposure to either basic or acidic conditions, initiates a β-elimination reaction, resulting in the cleavage of the phosphodiester backbone 3′ to the AP-site and the formation of an α,β-unsaturated aldehyde. Subsequent δ-elimination induces DNA strand cleavage 5′ to the AP-site resulting in the generation of a single-nucleotide gap in dsDNA or strand cleavage in ssDNA.

    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).

    Techniques: Activity Assay

    Representative sequence motifs for the UDG-mediated uracil cleavage on double- ( A , B ) and single-stranded ( C , D ) DNA strands. Substrates were incubated with UDG for different time periods ranging from 5 seconds to 30 minutes (since the cleavage motifs showed little to no sequence dependence, only the 5s, 30s, 60s and 120s were determined for ssDNA). The sequence motifs were extracted from the 1% (41 of 4096 sequences) most cleaved ( A , C ) and least cleaved ( B , D ) sequences of the library.

    Journal: Scientific Reports

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries

    doi: 10.1038/s41598-019-54044-x

    Figure Lengend Snippet: Representative sequence motifs for the UDG-mediated uracil cleavage on double- ( A , B ) and single-stranded ( C , D ) DNA strands. Substrates were incubated with UDG for different time periods ranging from 5 seconds to 30 minutes (since the cleavage motifs showed little to no sequence dependence, only the 5s, 30s, 60s and 120s were determined for ssDNA). The sequence motifs were extracted from the 1% (41 of 4096 sequences) most cleaved ( A , C ) and least cleaved ( B , D ) sequences of the library.

    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).

    Techniques: Sequencing, Incubation