uracil dna glycosylase udg  (New England Biolabs)


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

    New England Biolabs uracil dna glycosylase udg
    Uracil DNA Glycosylase UDG
    Uracil DNA Glycosylase UDG 5 000 units
    https://www.bioz.com/result/uracil dna glycosylase udg/product/New England Biolabs
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase udg - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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:

    6) 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

    7) 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:

    8) 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

    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