uracil dna glycosylase udg  (New England Biolabs)


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  • 99
    Name:
    Uracil DNA Glycosylase UDG
    Description:
    Uracil DNA Glycosylase UDG 5 000 units
    Catalog Number:
    M0280L
    Price:
    296
    Size:
    5 000 units
    Category:
    DNA Glycosylases
    Score:
    85
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    Structured Review

    New England Biolabs uracil dna glycosylase udg
    Uracil DNA Glycosylase UDG
    Uracil DNA Glycosylase UDG 5 000 units
    https://www.bioz.com/result/uracil dna glycosylase udg/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase udg - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) 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

    2) Product Images from "The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1053

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

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

    Related Articles

    Centrifugation:

    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: Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics
    Article Snippet: After cell debris was removed by centrifugation (13,000×g , 20 min) at 4°C, supernatant was collected and placed on ice. .. We purchased E. coli uracil-DNA glycosylase (UDG), formamidopyrimidine-DNA glycosylase (Fpg), endonuclease IV, and T4 polynucleotide kinase (New England Biolabs, UK).

    Amplification:

    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). .. 2.5 U/μL of Circligase II (Biozym 131406) was used and the ligation reaction carried out overnight.

    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: 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.

    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.
    E Coli Uracil Dna Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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