e coli uracil dna glycosylase  (New England Biolabs)


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    Name:
    Uracil DNA Glycosylase UDG
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    Uracil DNA Glycosylase UDG 5 000 units
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    M0280L
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    DNA Glycosylases
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    New England Biolabs e coli uracil dna glycosylase
    Uracil DNA Glycosylase UDG
    Uracil DNA Glycosylase UDG 5 000 units
    https://www.bioz.com/result/e coli uracil dna glycosylase/product/New England Biolabs
    Average 99 stars, based on 68 article reviews
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    e coli uracil dna glycosylase - by Bioz Stars, 2019-12
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    Images

    1) 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

    2) Product Images from "Timing Facilitated Site Transfer of an Enzyme on DNA"

    Article Title: Timing Facilitated Site Transfer of an Enzyme on DNA

    Journal: Nature Chemical Biology

    doi: 10.1038/nchembio.764

    Facilitated site transfer by human uracil DNA glycosylase (hUNG). In all facilitated transfer assays the hUNG concentration is 5–20 pM and the DNA substrate concentration is 40 nM. (a) Facilitated site transfer of hUNG between two uracil sites on the same DNA strand separated by 10 bps (S10). Reactions in the absence and presence of 10 mM uracil are shown. Facilitated transfer is qualitatively indicated by an excess of double excision fragments (A and C) relative single site excision products (AB and BC). (b) Facilitated site transfer by hUNG between sites separated by 5 bp (S5) in the absence and presence of 10 mM uracil. (c) Observed probability of site transfer (P trans obs ) as a function of time and uracil concentration for the substrate with a 10 bp site separation. Linear extrapolation to the y axis provides the true transfer probability at zero time (P trans ). (d) P trans ′ as a function of increasing uracil for substrates with 5, 10 and 20 bp site spacings. Each data point represents an individual experiment as in panels a and b and the prime notation indicates correction for the efficiency of excision (see text). The non-linear least squares fits in (d) use a kinetic partitioning model that relates the dependence of the total transfer probability (P trans ′ = P slide ′ + P hop ′) to the uracil trap concentration ( Supplementary Methods ). Uncut gel images are shown in Supplementary Fig. 6 . Error bars represent the mean ± 1 s.d. of at least three independent trials.
    Figure Legend Snippet: Facilitated site transfer by human uracil DNA glycosylase (hUNG). In all facilitated transfer assays the hUNG concentration is 5–20 pM and the DNA substrate concentration is 40 nM. (a) Facilitated site transfer of hUNG between two uracil sites on the same DNA strand separated by 10 bps (S10). Reactions in the absence and presence of 10 mM uracil are shown. Facilitated transfer is qualitatively indicated by an excess of double excision fragments (A and C) relative single site excision products (AB and BC). (b) Facilitated site transfer by hUNG between sites separated by 5 bp (S5) in the absence and presence of 10 mM uracil. (c) Observed probability of site transfer (P trans obs ) as a function of time and uracil concentration for the substrate with a 10 bp site separation. Linear extrapolation to the y axis provides the true transfer probability at zero time (P trans ). (d) P trans ′ as a function of increasing uracil for substrates with 5, 10 and 20 bp site spacings. Each data point represents an individual experiment as in panels a and b and the prime notation indicates correction for the efficiency of excision (see text). The non-linear least squares fits in (d) use a kinetic partitioning model that relates the dependence of the total transfer probability (P trans ′ = P slide ′ + P hop ′) to the uracil trap concentration ( Supplementary Methods ). Uncut gel images are shown in Supplementary Fig. 6 . Error bars represent the mean ± 1 s.d. of at least three independent trials.

    Techniques Used: Concentration Assay

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn118

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

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

    4) Product Images from "The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence"

    Article Title: The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku602

    Glycosylase repair diminishes as Pot1 binding affinity increases. Competition between Pot1pN binding and glycosylase activity on single-stranded oligonucleotides with pyrimidine substitutions at positions T 3 and T 4 is shown. ( a ) Uracil substitution and UNG cleavage. ( b ) 5FU substitution and UNG cleavage. ( c ) 5hmU substitution and hSMUG1 cleavage. Lane 1: negative control without the addition of Pot1pN or a glycosylase; Lane 2: positive control with the addition of a glycosylase; Lane 3: simultaneous incubation with both Pot1pN and a glycosylase.
    Figure Legend Snippet: Glycosylase repair diminishes as Pot1 binding affinity increases. Competition between Pot1pN binding and glycosylase activity on single-stranded oligonucleotides with pyrimidine substitutions at positions T 3 and T 4 is shown. ( a ) Uracil substitution and UNG cleavage. ( b ) 5FU substitution and UNG cleavage. ( c ) 5hmU substitution and hSMUG1 cleavage. Lane 1: negative control without the addition of Pot1pN or a glycosylase; Lane 2: positive control with the addition of a glycosylase; Lane 3: simultaneous incubation with both Pot1pN and a glycosylase.

    Techniques Used: Binding Assay, Activity Assay, Negative Control, Positive Control, Incubation

    5) 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

    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, Flow Cytometry

    7) Product Images from "Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues"

    Article Title: Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt625

    Identification of putative helix-invading residues in ROS1. ( A ) Schematic diagram showing conserved regions among members of the ROS1/DME family: a N-terminal lysine-rich region (green), a noncontiguous DNA glycosylase domain distributed over two segments (blue and red) separated by a nonstructured linker region and a highly conserved C-terminal domain (yellow) that is not found in any other protein family. ( B ) Multiple sequence alignment of part of the DNA glycosylase domain of ROS1/DME proteins and several HhH-GPD superfamily members. ROS1 amino acids analyzed in this work are indicated by inverted triangles and highlighted in orange (Q607), green (R903) or pink (M905). 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 as follows: 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 ) Structural model for the DNA glycosylase domain of ROS1 bound to a DNA containing an abasic site. The position of Q607, R903 and M905 residues (colored as in panel B) in relation to the estranged guanine (blue) are shown. The model was generated as described in ( 20 ) and the figure was prepared with PyMOL ( http://www.pymol.org ). ( D ) Schematic sequence diagram indicating the predicted interactions between mutated amino acids and the orphan guanine (blue) opposite 5-meC (M, in red).
    Figure Legend Snippet: Identification of putative helix-invading residues in ROS1. ( A ) Schematic diagram showing conserved regions among members of the ROS1/DME family: a N-terminal lysine-rich region (green), a noncontiguous DNA glycosylase domain distributed over two segments (blue and red) separated by a nonstructured linker region and a highly conserved C-terminal domain (yellow) that is not found in any other protein family. ( B ) Multiple sequence alignment of part of the DNA glycosylase domain of ROS1/DME proteins and several HhH-GPD superfamily members. ROS1 amino acids analyzed in this work are indicated by inverted triangles and highlighted in orange (Q607), green (R903) or pink (M905). 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 as follows: 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 ) Structural model for the DNA glycosylase domain of ROS1 bound to a DNA containing an abasic site. The position of Q607, R903 and M905 residues (colored as in panel B) in relation to the estranged guanine (blue) are shown. The model was generated as described in ( 20 ) and the figure was prepared with PyMOL ( http://www.pymol.org ). ( D ) Schematic sequence diagram indicating the predicted interactions between mutated amino acids and the orphan guanine (blue) opposite 5-meC (M, in red).

    Techniques Used: Sequencing, Generated

    Q607A, M905G and R903A mutant proteins lack DNA glycosylase activity on 5-meC:G pairs but retain AP liase activity. ( A ) Schematic diagram of ROS1 DNA glycosylase/AP lyase activity on 5-meC. ROS1 excises 5-meC as a free base and then cleaves the phosphodiester backbone at the 5-meC removal site by successive β,δ-elimination. ( B ) DNA glycosylase assay. The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 4 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. After incubation, NaOH (100 nM) was added and samples were 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. ( C ) AP liase assay. A double-stranded oligonucleotide substrate containing an AP site opposite G (20 nM) was incubated at 30°C for 2 h in the presence of purified WT ROS1 or mutant variants (20 nM). Samples were treated with NaBH 4 (300 mM) at 0°C for 30 min to stabilize nonprocessed AP sites and neutralized with 100 mM Tris–HCl, pH 7.4. Products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are mean ± SE (error bars) from three independent experiments.
    Figure Legend Snippet: Q607A, M905G and R903A mutant proteins lack DNA glycosylase activity on 5-meC:G pairs but retain AP liase activity. ( A ) Schematic diagram of ROS1 DNA glycosylase/AP lyase activity on 5-meC. ROS1 excises 5-meC as a free base and then cleaves the phosphodiester backbone at the 5-meC removal site by successive β,δ-elimination. ( B ) DNA glycosylase assay. The generation of incision products was measured by incubating purified WT ROS1 or mutant variants (20 nM) at 30°C for 4 h with a double-stranded oligonucleotide substrate (20 nM) containing a single 5-meC:G pair. After incubation, NaOH (100 nM) was added and samples were 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. ( C ) AP liase assay. A double-stranded oligonucleotide substrate containing an AP site opposite G (20 nM) was incubated at 30°C for 2 h in the presence of purified WT ROS1 or mutant variants (20 nM). Samples were treated with NaBH 4 (300 mM) at 0°C for 30 min to stabilize nonprocessed AP sites and neutralized with 100 mM Tris–HCl, pH 7.4. Products were separated in a 12% denaturing polyacrylamide gel and the amount of incised oligonucleotide was quantified by fluorescent scanning. Values are mean ± SE (error bars) from three independent experiments.

    Techniques Used: Mutagenesis, Activity Assay, Purification, Incubation

    8) Product Images from "Molecular crowding enhances facilitated diffusion of two human DNA glycosylases"

    Article Title: Molecular crowding enhances facilitated diffusion of two human DNA glycosylases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv301

    General schematic of how the introduction of molecular crowders (orange lines) can influence individual steps in the DNA glycosylase damage search pathway. These steps including the rate of diffusion to the DNA chain ( k on ), the lifetime of nonspecific (1/ k off N ) and specific (1/ k off S ) DNA complexes, the probability of associative and dissociative transfers between damage sites, and changes in the rate of product release (rate-limiting for k cat ). Dashed lines represent the sizes of the depletion layers surrounding the protein and DNA where the PEG 8K polymer, but not solv ent, is excluded. For large DNA molecules, association is limited by translation of the protein through the crowded solution ( k on bulk ) until their depletion layers overlap and association proceeds within a low viscosity environment. Also depicted are highly dynamic closed-to-open conformational changes in hUNG and hOGG1 that accompany nonspecific DNA binding ( 42 , 50 ); only the open state is viewed competent for translocation ( 50 ). Release of each enzyme from the product requires an even larger closed-to-open transition that has been shown to be at least partly rate-limiting for turnover of hUNG ( k′ open ) ( 46 ). Image is drawn to scale using a DNA duplex of 2 nm as a scale reference. The depletion layer size in the dilute regime for PEG 8K is shown (equivalent to R g PEG , see Supplemental Information).
    Figure Legend Snippet: General schematic of how the introduction of molecular crowders (orange lines) can influence individual steps in the DNA glycosylase damage search pathway. These steps including the rate of diffusion to the DNA chain ( k on ), the lifetime of nonspecific (1/ k off N ) and specific (1/ k off S ) DNA complexes, the probability of associative and dissociative transfers between damage sites, and changes in the rate of product release (rate-limiting for k cat ). Dashed lines represent the sizes of the depletion layers surrounding the protein and DNA where the PEG 8K polymer, but not solv ent, is excluded. For large DNA molecules, association is limited by translation of the protein through the crowded solution ( k on bulk ) until their depletion layers overlap and association proceeds within a low viscosity environment. Also depicted are highly dynamic closed-to-open conformational changes in hUNG and hOGG1 that accompany nonspecific DNA binding ( 42 , 50 ); only the open state is viewed competent for translocation ( 50 ). Release of each enzyme from the product requires an even larger closed-to-open transition that has been shown to be at least partly rate-limiting for turnover of hUNG ( k′ open ) ( 46 ). Image is drawn to scale using a DNA duplex of 2 nm as a scale reference. The depletion layer size in the dilute regime for PEG 8K is shown (equivalent to R g PEG , see Supplemental Information).

    Techniques Used: Diffusion-based Assay, Binding Assay, Translocation Assay

    9) 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:

    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

    10) 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

    11) Product Images from "Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases"

    Article Title: Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

    Journal:

    doi: 10.1074/jbc.M112.403253

    Collision-induced dissociation spectra from LC-MS/MS analysis of the full-length extension assays using pol ι and a 23-mer oligomer template containing 2′-F- N 2 ,3-ϵdG in the presence of all four dNTPs. A , template and primer sequences. The confirmed product sequences with the fragmentation patterns shown are 5′-CCATGA-3′ ( B ), 5′-CTATGA-3′ ( C ), 5′-CAATGA-3′ ( D ), 5′-CCATGAA-3′ ( E ), and 5′-CTATGAA-3′ ( F ). The reaction contained 12.5 μ m DNA complex, 10 m m dNTPs, 10 μ m pol ι, 5 m m DTT, 50 m m NaCl, 5 m m MgCl2 , and 50 μg ml−1 BSA and were incubated at 37 °C for 3.5 h. Underlined U indicates the cutting site by uracil-DNA glycosylase after DNA polymerase reactions.
    Figure Legend Snippet: Collision-induced dissociation spectra from LC-MS/MS analysis of the full-length extension assays using pol ι and a 23-mer oligomer template containing 2′-F- N 2 ,3-ϵdG in the presence of all four dNTPs. A , template and primer sequences. The confirmed product sequences with the fragmentation patterns shown are 5′-CCATGA-3′ ( B ), 5′-CTATGA-3′ ( C ), 5′-CAATGA-3′ ( D ), 5′-CCATGAA-3′ ( E ), and 5′-CTATGAA-3′ ( F ). The reaction contained 12.5 μ m DNA complex, 10 m m dNTPs, 10 μ m pol ι, 5 m m DTT, 50 m m NaCl, 5 m m MgCl2 , and 50 μg ml−1 BSA and were incubated at 37 °C for 3.5 h. Underlined U indicates the cutting site by uracil-DNA glycosylase after DNA polymerase reactions.

    Techniques Used: Liquid Chromatography, Mass Spectrometry, Incubation

    12) 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

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

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

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

    14) Product Images from "Arabidopsi"

    Article Title: Arabidopsi

    Journal:

    doi: 10.1074/jbc.M109.067173

    UDG activity is intrinsic to AtUNG. A , effect of Ugi on AtUNG activity. A DNA duplex (40 n m ) containing an U:G mispair was incubated with AtUNG (2 n m ) or E. coli Ung (1 unit) during 30 min at 30 °C in the absence or the presence of the peptide inhibitor Ugi (2 units). Lane 1 , control reaction without enzyme. Lane 6 , 28-nt marker. B , effect of D173N mutation on AtUNG activity. A DNA duplex (40 n m ) containing an U:G mispair was incubated with wild-type AtUNG (2 n m ) or with mutant AtUNG-D173N (2 or 50 n m ) during 30 min at 30 °C in the absence or the presence of the peptide inhibitor Ugi (2 units), as indicated. Reaction products were separated in a 12% denaturing polyacrylamide gel, and detected by fluorescence scanning. Lane 1 , 28-nt marker.
    Figure Legend Snippet: UDG activity is intrinsic to AtUNG. A , effect of Ugi on AtUNG activity. A DNA duplex (40 n m ) containing an U:G mispair was incubated with AtUNG (2 n m ) or E. coli Ung (1 unit) during 30 min at 30 °C in the absence or the presence of the peptide inhibitor Ugi (2 units). Lane 1 , control reaction without enzyme. Lane 6 , 28-nt marker. B , effect of D173N mutation on AtUNG activity. A DNA duplex (40 n m ) containing an U:G mispair was incubated with wild-type AtUNG (2 n m ) or with mutant AtUNG-D173N (2 or 50 n m ) during 30 min at 30 °C in the absence or the presence of the peptide inhibitor Ugi (2 units), as indicated. Reaction products were separated in a 12% denaturing polyacrylamide gel, and detected by fluorescence scanning. Lane 1 , 28-nt marker.

    Techniques Used: Activity Assay, Incubation, Marker, Mutagenesis, Fluorescence

    15) 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

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    Binding Assay:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Oligonucleotide Assay:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. Cleavage reactions were stopped by addition of 20 µl 0.4% fushin in formamide and denaturation at 90°C for 3 min. After quenching on ice, samples were resolved on 17.5% TBE-PAGE urea gels at 200 V and visualised using a Typhoon scanner (GE Healthcare) for fluorescence imaging (Filter: 526 SP (532 nm), Laser: Blue 488 nm).

    Synthesized:

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition
    Article Snippet: Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA). .. Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: The second-strand cDNA was synthesized with 100 mM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Construct:

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Deamination activity was measured using a labeled ssDNA construct 118 nt in length with potential deamination sites 17 nt from the 5′-end and 36 nt from the 3′-end (see Supplementary Fig. for full sequence). .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Real-time Polymerase Chain Reaction:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA.

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Incubation:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: Reactions were stopped and brought to 100 µl total volume in H2 O. .. 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. Cleavage reactions were stopped by addition of 20 µl 0.4% fushin in formamide and denaturation at 90°C for 3 min. After quenching on ice, samples were resolved on 17.5% TBE-PAGE urea gels at 200 V and visualised using a Typhoon scanner (GE Healthcare) for fluorescence imaging (Filter: 526 SP (532 nm), Laser: Blue 488 nm).

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: PAGE purified deoxyoligonucleotide ( ) was 5′-end 32 P labeled and added into 20 µl of deaminase reaction buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C. .. The samples were incubated at 95°C for 5 min, 4°C for 2 min and separated by 15% TBE/urea-PAGE.

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: After incubations for 1, 2, 5, and 10 min, the reactions were quenched by a double extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by a buffer exchange to 10 m m Tris (pH 7.4) using a Micro Bio-spin P-6 column (Bio-Rad). .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad). .. Products of single deamination at the 5′-side (5′), the 3′-side (3′) target, or double deaminations at both sites (5′ and 3′) were detected as 54, 43, and 32 nt cleaved products, respectively.

    Article Title: Transient-state kinetics of apurinic/apyrimidinic (AP) endonuclease 1 acting on an authentic AP site and commonly-used substrate analogs: The effect of diverse metal ions and base mismatches
    Article Snippet: Assembly of the 30-mer duplex substrate was achieved by annealing 30 pmol (for transient-state experiments) or 300 pmol (for steady-state experiments) of the 5′-radiolabeled lesion-containing strand in the presence of a 1.5-fold excess of the desired complement [either well matched (WM) or containing a mismatch (MM1, MM2, MM3, or MM4)] in 300 μL of 50 mM HEPES-KOH, 100 mM KCl, pH 7.5. .. To generate a duplex containing the reduced AP site, the uracil-containing duplex was incubated with 1.5 units of uracil DNA glycosylase (UDG; New England Biolabs) and freshly prepared NaBH4 (final concentration of NaBH4 was 0.1 M) overnight at 37 °C. .. The DNA was desalted using a 0.5 mL, 3,000 MW Amicon centrifugal filter.

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: The labeled DNA was then added at a concentration of 100 nM and incubated for 10 min at 37 °C. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. This treatment fragmented the ssDNA at deamination sites.

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK). .. We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The PCR cycling and melting conditions were as follows; an initial incubation at 95°C for 15 min, followed by 55 cycles of 96°C for 15 s, 70°C for 20 s, 72°C for 30 s; one cycle of 97°C for 1 min and a melt from 70°C to 95°C rising 0.2°C per second. .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The reaction mixture in a final volume of 20 μL was prepared as follows; 1 × PCR buffer, 2.5 mM MgCl2, 400 nM of each primer, 10 ng of FFPE DNA, 200 μM of dNTPs, 5 μM of SYTO9 (Invitrogen), and 0.5 U of HotStarTaq polymerase (Qiagen). .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Activity Assay:

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: Paragraph title: Deamination Assay to Measure Apo3A Specific Activity on ssDNA ... Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Article Title: Specific Inhibition of NEIL-initiated Repair of Oxidized Base Damage in Human Genome by Copper and Iron
    Article Snippet: Paragraph title: Measurement of AP Lyase Activity ... After annealing with the complementary strand, the duplex was treated with 100 units of uracil-DNA glycosylase (New England Biolabs) at 37 °C for 30 min to generate an AP site by excising U.

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Modification:

    Article Title: DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site
    Article Snippet: The integrity of the AP-containing ODN was assessed by PAGE followed by Stains-All staining. .. To confirm the presence of the AP site in the ODN after the treatment with uracil-DNA glycosylase, samples were treated with 10% aqueous piperidine at 95 °C and were completely cleaved at the modified site. .. When needed, the modified strands were 32 P-labeled using [γ-32 P]ATP and phage T4 polynucleotide kinase (SibEnzyme, Novosibirsk, Russia) according to the manufacturer's protocol, purified by 20% denaturing PAGE, and annealed to the complementary strand.

    Hybridization:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    High Performance Liquid Chromatography:

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition
    Article Snippet: Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA). .. Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA).

    Flow Cytometry:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: The lysate-antibody was then incubated with High flow protein-G-Sepharose for 1-2 h at 4°C. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Chromatography:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: .. Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    Ligation:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Buffer Exchange:

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: After incubations for 1, 2, 5, and 10 min, the reactions were quenched by a double extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by a buffer exchange to 10 m m Tris (pH 7.4) using a Micro Bio-spin P-6 column (Bio-Rad). .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Transferring:

    Article Title: Telomere Restriction Fragment (TRF) Analysis
    Article Snippet: .. Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 ) 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 ) DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 ) Proteinase K (QIAGEN, catalog number: 19131 or 19133 ) Enzymes HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L ) HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L ) MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S ) HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L ) RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L ) AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L ) NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S) Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S ) Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S ) DEPC-treated water (Life Technologies, catalog number: AM9906 ) Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9906 ”. .. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 ) Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 ) Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 ) Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 ) Boric acid (Sigma-Aldrich, catalog number: B6768 ) UltraPure™Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 16500-500 ) GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 ) Radiolabelled TRF Marker ( ) DNA marker (Bionexus, catalog number: BN2050 ) Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 ) Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 ) Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 ) Polyvinylpyrolidone (Sigma-Aldrich, catalog number: PVP40 ) Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P ) dCTP, [α-32 P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 μCi (PerkinElmer, catalog number: NEG513Z500UC ) 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, Invitrogen™, catalog number: 15557-036 ) Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 ) 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 ) Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001 ”.

    Generated:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: We interpret this result as indication that no obvious negative influence on extraction efficiency is caused by using this reduced input bone powder amount. .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. The Klenow Fragment of DNA polymerase I (Thermo Fisher Scientific EP0051) was used for the fill-in reaction [ ].

    Article Title: Specific Inhibition of NEIL-initiated Repair of Oxidized Base Damage in Human Genome by Copper and Iron
    Article Snippet: We generated an AP site-containing duplex oligonucleotide substrate starting with the 51-nt oligonucleotide as described earlier, except for incorporation of U at position 26. .. After annealing with the complementary strand, the duplex was treated with 100 units of uracil-DNA glycosylase (New England Biolabs) at 37 °C for 30 min to generate an AP site by excising U.

    other:

    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).

    Polymerase Chain Reaction:

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: Primers labeled with Cy5, Cy3, or 6-FAM (6-carboxyfluorescein) were used for PCR. .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). .. Completed libraries were quantified with the Agilent Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS Library Quantification kit.

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK). .. Micro Bio-Spin 30 Chromatography Columns were from Bio-Rad.

    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: Oligonucleotides for PCR primers and substrates containing uracil (U) were purchased from IDT (Coralville, IA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The PCR cycling and melting conditions were as follows; an initial incubation at 95°C for 15 min, followed by 55 cycles of 96°C for 15 s, 70°C for 20 s, 72°C for 30 s; one cycle of 97°C for 1 min and a melt from 70°C to 95°C rising 0.2°C per second. .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix. .. The same PCR conditions were used except an addition of initial incubation at 37°C for 30 min before the activation of HotStarTaq polymerase.

    Sonication:

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: The cell pellet was resuspended in < 5 ml of sonication buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.1 mM DTT), and cells were lysed by sonification. .. We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Recombinant:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: Oligonucleotides were incubated for 30 min at 37°C with 50 ng of recombinant protein. .. 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. All DNAs, with and without gap, were PAGE-purified using a Mini Prep Cell (Bio-Rad, Hercules, CA).

    Article Title: Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene
    Article Snippet: Recombinant human DNA polymerases hPol η (amino acids 1–437), hPol ι (amino acids 1–420), and hPol κ (amino acids 19–526) (active core enzymes) were expressed and purified according to previously published procedures. .. – T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA).

    DNA Extraction:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: DNA extraction was performed according to Dabney et al. [ ] with reduced bone powder input mass, and reduced centrifugation speed of the binding apparatus at approximately 450×g . .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299).

    Nucleic Acid Electrophoresis:

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Article Title: Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene
    Article Snippet: Full-length recombinant human polymerase κ (hPol κ ) used for gel electrophoresis experiments was purchased from Enzymax (Lexington, KY). .. – T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA).

    RNA Sequencing Assay:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). .. Completed libraries were quantified with the Agilent Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS Library Quantification kit.

    Fluorescence:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA.

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: After incubations for 1, 2, 5, and 10 min, the reactions were quenched by a double extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by a buffer exchange to 10 m m Tris (pH 7.4) using a Micro Bio-spin P-6 column (Bio-Rad). .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad). .. Products of single deamination at the 5′-side (5′), the 3′-side (3′) target, or double deaminations at both sites (5′ and 3′) were detected as 54, 43, and 32 nt cleaved products, respectively.

    Magnetic Beads:

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: Reactions were stopped and brought to 100 µl total volume in H2 O. .. 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. Cleavage reactions were stopped by addition of 20 µl 0.4% fushin in formamide and denaturation at 90°C for 3 min. After quenching on ice, samples were resolved on 17.5% TBE-PAGE urea gels at 200 V and visualised using a Typhoon scanner (GE Healthcare) for fluorescence imaging (Filter: 526 SP (532 nm), Laser: Blue 488 nm).

    Mutagenesis:

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: Paragraph title: Mutation profiling by amplicon sequencing ... For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Isolation:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Labeling:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: PAGE purified deoxyoligonucleotide ( ) was 5′-end 32 P labeled and added into 20 µl of deaminase reaction buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: Primers labeled with Cy5, Cy3, or 6-FAM (6-carboxyfluorescein) were used for PCR. .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ).

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: For reactions at pH 5.1, 5.5, and 6.5, 500 n m labeled ssDNA was incubated with 5 n m Apo3A in the reaction buffer (30 μl) at 37 °C. .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: The labeled DNA was then added at a concentration of 100 nM and incubated for 10 min at 37 °C. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Purification:

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition
    Article Snippet: Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA). .. Uracil DNA glycosylase (UDG), λ exonuclease (λexo ) and hoGG I were obtained from New England Biolabs (Beverly, MA, USA).

    Article Title: Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways
    Article Snippet: 8 µl of streptavidin magnetic beads (Dynal M270, Invitrogen) were incubated with the oligonucleotides in TE-1000 for 15 min. Beads were collected, washed twice in TE-1000 preheated to 70°C, once in TE at RT, and resuspended in Uracil-DNA Glycosylase (UDG, New England Biolabs) reaction mix, prepared according to manufacturer protocol, and incubated for 1 h at 37°C. .. The UNG oligonucleotide assay was performed with oligonucleotides 164 (5′-B–ATTATTATTATTAGUTATTTATTTATTTATTTATTTATTT-FITC-3′) and 166 ( 5′-AAATAAATAAATAAATAAATAAATAGCTAATAATAATAAT-3′ ).

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: PAGE purified deoxyoligonucleotide ( ) was 5′-end 32 P labeled and added into 20 µl of deaminase reaction buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. All DNAs, with and without gap, were PAGE-purified using a Mini Prep Cell (Bio-Rad, Hercules, CA).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Article Title: Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene
    Article Snippet: Recombinant human DNA polymerases hPol η (amino acids 1–437), hPol ι (amino acids 1–420), and hPol κ (amino acids 19–526) (active core enzymes) were expressed and purified according to previously published procedures. .. – T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA).

    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Sequencing:

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: We interpret this result as indication that no obvious negative influence on extraction efficiency is caused by using this reduced input bone powder amount. .. Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. The Klenow Fragment of DNA polymerase I (Thermo Fisher Scientific EP0051) was used for the fill-in reaction [ ].

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Deamination activity was measured using a labeled ssDNA construct 118 nt in length with potential deamination sites 17 nt from the 5′-end and 36 nt from the 3′-end (see Supplementary Fig. for full sequence). .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: Paragraph title: Mutation profiling by amplicon sequencing ... For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: PAGE purified deoxyoligonucleotide ( ) was 5′-end 32 P labeled and added into 20 µl of deaminase reaction buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: After incubations for 1, 2, 5, and 10 min, the reactions were quenched by a double extraction with phenol:chloroform:isoamyl alcohol (25:24:1) followed by a buffer exchange to 10 m m Tris (pH 7.4) using a Micro Bio-spin P-6 column (Bio-Rad). .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad). .. Products of single deamination at the 5′-side (5′), the 3′-side (3′) target, or double deaminations at both sites (5′ and 3′) were detected as 54, 43, and 32 nt cleaved products, respectively.

    IA:

    Article Title: Characterization of the 3' to 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues
    Article Snippet: Oligonucleotides for PCR primers and substrates containing uracil (U) were purchased from IDT (Coralville, IA). .. E. coli uracil DNA glycosylase (UDG), and restriction enzymes were purchased from New England Biolabs (Beverly, MA).

    Mouse Assay:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    HRM Assay:

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix. .. A PIK3CA E549D mutation detected at a low frequency (4%) in the Core 2 of Tumor 2 by AmpliSeq was analysed by limited copy number (LCN)-HRM [ ].

    Plasmid Preparation:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C. .. For in vitro synthesis of A3A and mutants we employed the TNT Coupled Wheat Germ Extract System (Promega) using T7 polymerase.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: DNA for nucleosome preparations was PCR amplified from a plasmid containing the Widom's 601 DNA ( ). .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ).

    Software:

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl.

    SYBR Green Assay:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA. .. Seal the plate with a plastic seal (Bio-Rad catalog# MSB1001), and use the following PCR protocol: 37°C 10 min 95°C 10 min 95°C 30 s 60°C 30 s 74°C 10 s Read plate.

    Article Title: Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone
    Article Snippet: Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with uracil-DNA glycosylase (New England Biolabs M0279) and endonuclease VIII (New England Biolabs M0299). .. A quantitative PCR (qPCR) experiment was carried out using 0.2% of the unamplified library to estimate relative library complexities (Additional file : Table S1), and to determine the optimal number of cycles for subsequent indexing PCR, representing the inflection point of the respective library amplification curves, corrected for reaction volume and template amount. qPCR was performed on a PikoReal 96 Real-Time PCR machine (Thermo Fisher Scientific TCR0096) with 3 replicates for each library, involving an initial 10 min denaturation at 95 °C, followed by 40 cycles of: 15 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C.

    Selection:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    In Vitro:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: Paragraph title: In vitro Deaminase Assays ... The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Article Title: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: Paragraph title: In vitro base-excision repair (BER) assay ... We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Irradiation:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Test your dilution series for linearity in a real-time PCR machine with a fluorescence detector (Bio-Rad Opticon 2, catalog# CFB-3120 or comparable machine) using the control primers (Forward: 5′-CTCACCAAAAACAAAAACAGCC-3′, Reverse: 5′-CTTTTGTCCCTCCCACTTTGG-3′). .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA. .. Seal the plate with a plastic seal (Bio-Rad catalog# MSB1001), and use the following PCR protocol: 37°C 10 min 95°C 10 min 95°C 30 s 60°C 30 s 74°C 10 s Read plate.

    Produced:

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Concentration Assay:

    Article Title: A random mutation capture assay to detect genomic point mutations in mouse tissue
    Article Snippet: Store your dilutions at 4°C until validated as freeze–thaw cycles alter the effective DNA concentration. .. For each well in a 96-well plate (Bio-Rad catalog# HSP9655), add 12.5 µl SYBR green master mix (Applied Biosystems catalog# 4309155), 0.5 µl Uracil–DNA glycosylase (UDG, New England Biolabs catalog# M0280L), 0.5 µl of 15 µM stock of control primers (Fwd and Rev), 6.5 µl irradiated water and 5 µl of diluted DNA.

    Article Title: A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer
    Article Snippet: For reactions at pH 7.4 and 8.0, a higher concentration (50 n m ) of Apo3A was used. .. Deaminated products were treated with 1.5 units of uracil:DNA:glycosylase (New England Biolabs) for 30 min at 37 °C followed by incubation at 95 °C for 5 min in the presence of 0.1 n NaOH to cleave the abasic sites resulting from the removal of U. Cleaved deaminated products were separated on a 16% denaturing PAGE gel, visualized, and quantified using an FX fluorescence scanner (Bio-Rad).

    Article Title: Transient-state kinetics of apurinic/apyrimidinic (AP) endonuclease 1 acting on an authentic AP site and commonly-used substrate analogs: The effect of diverse metal ions and base mismatches
    Article Snippet: Assembly of the 30-mer duplex substrate was achieved by annealing 30 pmol (for transient-state experiments) or 300 pmol (for steady-state experiments) of the 5′-radiolabeled lesion-containing strand in the presence of a 1.5-fold excess of the desired complement [either well matched (WM) or containing a mismatch (MM1, MM2, MM3, or MM4)] in 300 μL of 50 mM HEPES-KOH, 100 mM KCl, pH 7.5. .. To generate a duplex containing the reduced AP site, the uracil-containing duplex was incubated with 1.5 units of uracil DNA glycosylase (UDG; New England Biolabs) and freshly prepared NaBH4 (final concentration of NaBH4 was 0.1 M) overnight at 37 °C. .. The DNA was desalted using a 0.5 mL, 3,000 MW Amicon centrifugal filter.

    Article Title: Dimerization regulates both deaminase-dependent and deaminase-independent HIV-1 restriction by APOBEC3G
    Article Snippet: The labeled DNA was then added at a concentration of 100 nM and incubated for 10 min at 37 °C. .. Reactions were stopped by phenol:chloroform extraction and formation of uracils was detected by incubation of ssDNA with Uracil DNA N -glycosylase (New England Biolabs) and heating in the presence of NaOH.

    Lysis:

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase
    Article Snippet: The resin was washed three times with lysis buffer. .. The supernatant was incubated with uracil DNA glycosylase (New England Biolabs) in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C.

    Variant Assay:

    Article Title: A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine
    Article Snippet: Fluorescein-labeled DNA was visualized in a FLA-5100 imager and analyzed using Multigauge software (Fujifilm). .. When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl. .. Reactions were stopped by adding 20 mM EDTA, 0.6% sodium dodecyl sulphate, and 0.5 mg/ml proteinase K. After adding 10 μl of 90% formamide, samples were heated at 95°C for 5 min. Products were resolved and analyzed as described above.

    Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors
    Article Snippet: The HRM primer sequences for FGFR3 exon 6 variant analysis were F 5′-CAGTGGCGGTGGTGGTGAGG-3′ and R 5′-ACCTTGCAGTGGAACTCCACGTC-3′. .. For UDG treatment, 0.5 × UDG buffer and 0.5 U of uracil-DNA glycosylase (New England Biolabs) were added to the PCR master mix.

    Gas Chromatography-Mass Spectrometry:

    Article Title: Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine, and DNA uracil concentrations
    Article Snippet: Specifically, persons taking certain antipurines (azathioprine), chemotherapy drugs (methotrexate), or antibiotics (sulfamethoxazole and trimethoprim) were excluded. .. DNA was extracted from 1 mL whole frozen blood by using standard phenol:chloroform extraction after proteinase K and RNase treatment; 5.0–10 μ g DNA was used to determine the uracil content after uracil DNA glycosylase (New England Biolabs, Ipswich, MA) treatment according to the gas chromatography–mass spectrometry method of Blount and Ames ( ) with modifications ( ). .. Sufficient DNA for uracil analysis was recovered from 255 individuals.

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    New England Biolabs e coli uracil dna glycosylase
    GO <t>glycosylase/lyase</t> activity assay of putative TvoOgg. (a) Time course of TvoOgg glycosylase activity on substrate ∗ GO/C. 34-bp heteroduplex <t>DNA</t> 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.
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    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.

    Journal: Archaea

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

    doi: 10.1155/2016/8734894

    Figure Lengend 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.

    Article Snippet: Ten pmol of double-stranded DNA containing a U/C mismatch was incubated with 1.25 U of E. coli uracil DNA glycosylase (UDG, New England BioLabs, UK), in the reaction buffer for UDG, for 30 min at 37°C.

    Techniques: 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.

    Journal: Archaea

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

    doi: 10.1155/2016/8734894

    Figure Lengend 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.

    Article Snippet: Ten pmol of double-stranded DNA containing a U/C mismatch was incubated with 1.25 U of E. coli uracil DNA glycosylase (UDG, New England BioLabs, UK), in the reaction buffer for UDG, for 30 min at 37°C.

    Techniques: 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.

    Journal: Archaea

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

    doi: 10.1155/2016/8734894

    Figure Lengend 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.

    Article Snippet: Ten pmol of double-stranded DNA containing a U/C mismatch was incubated with 1.25 U of E. coli uracil DNA glycosylase (UDG, New England BioLabs, UK), in the reaction buffer for UDG, for 30 min at 37°C.

    Techniques: 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.

    Journal: Archaea

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

    doi: 10.1155/2016/8734894

    Figure Lengend 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.

    Article Snippet: Ten pmol of double-stranded DNA containing a U/C mismatch was incubated with 1.25 U of E. coli uracil DNA glycosylase (UDG, New England BioLabs, UK), in the reaction buffer for UDG, for 30 min at 37°C.

    Techniques: Sequencing

    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.

    Journal: Carcinogenesis

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

    doi: 10.1093/carcin/bgp118

    Figure Lengend 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.

    Article Snippet: The U:G substrate was treated with uracil DNA glycosylase (New England Biolabs) to make the apurinic/apyrimidinic substrate.

    Techniques: 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.

    Journal: Carcinogenesis

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

    doi: 10.1093/carcin/bgp118

    Figure Lengend 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.

    Article Snippet: The U:G substrate was treated with uracil DNA glycosylase (New England Biolabs) to make the apurinic/apyrimidinic substrate.

    Techniques: 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.

    Journal: Carcinogenesis

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

    doi: 10.1093/carcin/bgp118

    Figure Lengend 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.

    Article Snippet: The U:G substrate was treated with uracil DNA glycosylase (New England Biolabs) to make the apurinic/apyrimidinic substrate.

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

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkq982

    Figure Lengend 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.

    Article Snippet: When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl.

    Techniques: 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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkq982

    Figure Lengend 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

    Article Snippet: When measuring AP lyase activity, a fluorescein-labeled oligonucleotide duplex containing U opposite G (200 nM) was incubated at 30°C for the indicated times in a reaction mixture containing 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 2.5 U of E. coli Uracil DNA glycosylase (New England BioLabs), and the indicated amounts of WT ROS1 or mutant variant in a total volume of 5 μl.

    Techniques: Activity Assay, Purification, Mutagenesis, Incubation

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw054

    Figure Lengend 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.

    Article Snippet: TdT, T4PNK, human AP endonuclease I (h APE1), E. coli Uracil DNA Glycosylase (UDG) and E. coli EndoIII, were from New England Biolabs.

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw054

    Figure Lengend 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.

    Article Snippet: TdT, T4PNK, human AP endonuclease I (h APE1), E. coli Uracil DNA Glycosylase (UDG) and E. coli EndoIII, were from New England Biolabs.

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