uracil dna glycosylase  (New England Biolabs)


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    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
    Uracil DNA Glycosylase UDG
    Uracil DNA Glycosylase UDG 5 000 units
    https://www.bioz.com/result/uracil dna glycosylase/product/New England Biolabs
    Average 99 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma"

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

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgp118

    Analysis of hOGG1 variants. ( A ) 8oxoG DNA glycosylase activity of S31P compared with WT hOGG1. A total of 3 and 10 ng enzymes were incubated with an 8oxoG:C oligonucleotide at 37°C for 30 min before cleavage of the phosphodiester backbone by NaOH. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand and C = cleavage product). ( B ) DNA binding properties of hOGG1 WT and S31P. WT and S31P hOGG1 (10, 30 and 100 ng) were incubated with 8oxoG:C DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.
    Figure Legend Snippet: Analysis of hOGG1 variants. ( A ) 8oxoG DNA glycosylase activity of S31P compared with WT hOGG1. A total of 3 and 10 ng enzymes were incubated with an 8oxoG:C oligonucleotide at 37°C for 30 min before cleavage of the phosphodiester backbone by NaOH. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand and C = cleavage product). ( B ) DNA binding properties of hOGG1 WT and S31P. WT and S31P hOGG1 (10, 30 and 100 ng) were incubated with 8oxoG:C DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.

    Techniques Used: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay

    Analysis of hMYH variants. ( A ) Adenine DNA glycosylase activities of hMYH WT, R260Q, H434D and S501F variants were measured by incubating the respective proteins (18 ng) with a duplex oligodeoxyribonucleotide containing a single A:8oxoG or A:G basepair at 37°C for 30 min. Strand cleavage after NaOH treatment was analyzed by 20% polyacrylamide gel electrophoresis and phosphorimaging (I = intact strand and C = cleavage product). ( B ) Different amounts (0.6–240 ng) of hMYH WT (□), R260Q (▴), H434D (X) and S501F (*) were assayed for A:8oxoG DNA glycosylase activities and percentage strand cleavage quantified with ImageQuant. Extract from Escherichia coli cells expressing empty vector and purified similarly as hMYH was used to measure the background level (⧫). ( C ) DNA binding properties of hMYH WT, R260Q, H434D and S501F (24 ng) to substrates containing A:8oxoG (left panel) or A:G (right panel). After incubation on ice, DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.
    Figure Legend Snippet: Analysis of hMYH variants. ( A ) Adenine DNA glycosylase activities of hMYH WT, R260Q, H434D and S501F variants were measured by incubating the respective proteins (18 ng) with a duplex oligodeoxyribonucleotide containing a single A:8oxoG or A:G basepair at 37°C for 30 min. Strand cleavage after NaOH treatment was analyzed by 20% polyacrylamide gel electrophoresis and phosphorimaging (I = intact strand and C = cleavage product). ( B ) Different amounts (0.6–240 ng) of hMYH WT (□), R260Q (▴), H434D (X) and S501F (*) were assayed for A:8oxoG DNA glycosylase activities and percentage strand cleavage quantified with ImageQuant. Extract from Escherichia coli cells expressing empty vector and purified similarly as hMYH was used to measure the background level (⧫). ( C ) DNA binding properties of hMYH WT, R260Q, H434D and S501F (24 ng) to substrates containing A:8oxoG (left panel) or A:G (right panel). After incubation on ice, DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Expressing, Plasmid Preparation, Purification, Binding Assay, Incubation

    Analysis of NEIL1 variants. ( A ) DNA glycosylase activity of G83D compared with WT NEIL1. Enzymes (2, 5, 10 and 20 ng) were incubated with different oligonucleotide substrates as indicated at 37°C for 30 min. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand, C = cleavage product, β = β elimination, δ = δ elimination cleavage, ss = single strand). ( B ) FaPy DNA glycosylase activity of NEIL1 WT (⧫) and G83D (▪). Enzymes (3, 10, 30 and 100 ng) were assayed for removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC). ( C ) DNA binding properties of NEIL1 WT and G83D. NEIL1 WT and G83D (20, 50 and 100 ng) were incubated with 5ohC:G DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein. ( D ) Nuclear localization of NEIL1 G83D and E181K. Asynchronous growing HeLa S3 cells were transiently transfected with constructs expressing NEIL1-EGFP, NEIL1G83D-EGFP or NEIL1E181K-EGFP. Cells were imaged directly by fluorescence microscopy for EGFP detection. DNA was stained with Hoechst 33342.
    Figure Legend Snippet: Analysis of NEIL1 variants. ( A ) DNA glycosylase activity of G83D compared with WT NEIL1. Enzymes (2, 5, 10 and 20 ng) were incubated with different oligonucleotide substrates as indicated at 37°C for 30 min. The reaction products were separated by 20% polyacrylamide gel electrophoresis and visualized by phosphorimaging. (I = intact strand, C = cleavage product, β = β elimination, δ = δ elimination cleavage, ss = single strand). ( B ) FaPy DNA glycosylase activity of NEIL1 WT (⧫) and G83D (▪). Enzymes (3, 10, 30 and 100 ng) were assayed for removal of faPy from [ 3 H]-methyl-faPy-poly(dG·dC). ( C ) DNA binding properties of NEIL1 WT and G83D. NEIL1 WT and G83D (20, 50 and 100 ng) were incubated with 5ohC:G DNA on ice and DNA–protein complexes (B = bound substrate) were separated from free DNA (F) by 10% native polyacrylamide gel electrophoresis. Control lanes were without addition of protein. ( D ) Nuclear localization of NEIL1 G83D and E181K. Asynchronous growing HeLa S3 cells were transiently transfected with constructs expressing NEIL1-EGFP, NEIL1G83D-EGFP or NEIL1E181K-EGFP. Cells were imaged directly by fluorescence microscopy for EGFP detection. DNA was stained with Hoechst 33342.

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn118

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn118

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

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

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

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

    Journal:

    doi: 10.1074/jbc.M112.403253

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

    Techniques Used: Liquid Chromatography, Mass Spectrometry, Incubation

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

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

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

    6) Product Images from "Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases"

    Article Title: Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases

    Journal:

    doi: 10.1016/j.dnarep.2013.09.004

    Role of the void-filling Met72 and adjacent His73 in lesion excision. Glycosylase assays with double-stranded Sp1:C (A) and ssSp1 (B) where the DNA substrate (20 nM) was combined with 16 nM of either WT or mutant MvNei2. WT MvNei2 is displayed as circles.
    Figure Legend Snippet: Role of the void-filling Met72 and adjacent His73 in lesion excision. Glycosylase assays with double-stranded Sp1:C (A) and ssSp1 (B) where the DNA substrate (20 nM) was combined with 16 nM of either WT or mutant MvNei2. WT MvNei2 is displayed as circles.

    Techniques Used: Mutagenesis

    7) Product Images from "Modulation of the Processive Abasic Site Lyase Activity of a Pyrimidine Dimer Glycosylase"

    Article Title: Modulation of the Processive Abasic Site Lyase Activity of a Pyrimidine Dimer Glycosylase

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2011.07.015

    Substrate design and computer modeling of processive versus distributive enzyme reactions (A) Schematic diagram of the design, construction, and utilization of DNAs containing closely positioned AP sites. The substrate 46-mer was constructed from the ligation of an unlabeled 16-mer containing a uracil (U) at position 10 from the 5′ end with a 32 P-labeled 30-mer containing a U at position 11 from the 5′ end of the 30-mer, using a scaffold 41-mer. The resulting 32 P-labeled 46-mer was purified and annealed with a 5-fold molar excess of complementary 46-mer. The duplex DNA was digested with uracil DNA glycosylase to create 2 AP sites in the labeled strand in which incision at only the 5′ AP site (site 1) yielded a labeled 36-mer, while incision at the 3′ AP site (site 2) yielded a labeled 28-mer. Dual incisions generated a labeled 18-mer. The appearance of two bands at the 18-mer position is characteristic of T4-pdg catalyzing a β-elimination reaction that is followed by a δ-elimination reaction. (B) Computer simulation of a kinetic nicking reaction in which nicking at either of the two AP sites is completely random (or haphazard). The percentage of DNA molecules with no incisions exponentially decays with a concomitant increase in DNA molecules that contain one incision (at either the 5′ or 3′ AP site, site 1 or 2, respectively). For a completely random incision mechanism, the theoretical maximum percentage of DNA molecules containing only one incision reaches ~50% and begins to decay. The accumulation of DNA molecules containing dual incisions displayed a characteristic lag, followed by a sigmoidal increase as total number of incisions increased. The reaction models a maximally distributive incision reaction. (C) Computer simulation of a kinetic nicking reaction in which every DNA-enzyme encounter results in incision at both AP sites. The exponential decrease in the no incision population is reciprocally matched by an increase in dually-incised DNA molecules. This simulation represents a maximally processive reaction. (D) Computer simulations of the accumulation of single-incised DNA molecules in which the probability of producing dual incisions prior to enzyme-DNA dissociation was varied between 0 to 80% of the encounters. The “no incision” and “dual incision” populations were omitted for clarity. Computer simulation of the reaction mechanism as it shifts from a random (distributive) mechanism toward a more processive incision reveals both that the magnitude of the percent DNA that accumulates with only one incision decreases and the time at which it is maximal occurs earlier in the reaction.
    Figure Legend Snippet: Substrate design and computer modeling of processive versus distributive enzyme reactions (A) Schematic diagram of the design, construction, and utilization of DNAs containing closely positioned AP sites. The substrate 46-mer was constructed from the ligation of an unlabeled 16-mer containing a uracil (U) at position 10 from the 5′ end with a 32 P-labeled 30-mer containing a U at position 11 from the 5′ end of the 30-mer, using a scaffold 41-mer. The resulting 32 P-labeled 46-mer was purified and annealed with a 5-fold molar excess of complementary 46-mer. The duplex DNA was digested with uracil DNA glycosylase to create 2 AP sites in the labeled strand in which incision at only the 5′ AP site (site 1) yielded a labeled 36-mer, while incision at the 3′ AP site (site 2) yielded a labeled 28-mer. Dual incisions generated a labeled 18-mer. The appearance of two bands at the 18-mer position is characteristic of T4-pdg catalyzing a β-elimination reaction that is followed by a δ-elimination reaction. (B) Computer simulation of a kinetic nicking reaction in which nicking at either of the two AP sites is completely random (or haphazard). The percentage of DNA molecules with no incisions exponentially decays with a concomitant increase in DNA molecules that contain one incision (at either the 5′ or 3′ AP site, site 1 or 2, respectively). For a completely random incision mechanism, the theoretical maximum percentage of DNA molecules containing only one incision reaches ~50% and begins to decay. The accumulation of DNA molecules containing dual incisions displayed a characteristic lag, followed by a sigmoidal increase as total number of incisions increased. The reaction models a maximally distributive incision reaction. (C) Computer simulation of a kinetic nicking reaction in which every DNA-enzyme encounter results in incision at both AP sites. The exponential decrease in the no incision population is reciprocally matched by an increase in dually-incised DNA molecules. This simulation represents a maximally processive reaction. (D) Computer simulations of the accumulation of single-incised DNA molecules in which the probability of producing dual incisions prior to enzyme-DNA dissociation was varied between 0 to 80% of the encounters. The “no incision” and “dual incision” populations were omitted for clarity. Computer simulation of the reaction mechanism as it shifts from a random (distributive) mechanism toward a more processive incision reveals both that the magnitude of the percent DNA that accumulates with only one incision decreases and the time at which it is maximal occurs earlier in the reaction.

    Techniques Used: Construct, Ligation, Labeling, Purification, Generated

    8) Product Images from "Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair"

    Article Title: Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair

    Journal:

    doi: 10.1073/pnas.042700399

    Preparation of site-specific DNA–protein crosslinks. ( A ) Sequence of the uracil-containing 60-mer oligodeoxynucleotide. ( B ) Urea-PAGE showing DNA substrate preparation. Lane 1, uracil-containing 60-mer; lane 2, uracil-containing 60-mer, digested with uracil DNA glycosylase and tested with T4-pdg (control of AP-site formation); reduced AP site-containing DNA before (lane 3) and after (lanes 4–6) purification; DPC-containing DNA before (lane 7) and after (lanes 8–10) purification. After purification, DNAs were subjected to the restriction endonuclease digestion with Sna BI ( , ) or Hae III ( , ). ( C ) SDS/PAGE showing DPC-containing DNA substrates before (lane 1) and after (lane 2) Hae III digestion.
    Figure Legend Snippet: Preparation of site-specific DNA–protein crosslinks. ( A ) Sequence of the uracil-containing 60-mer oligodeoxynucleotide. ( B ) Urea-PAGE showing DNA substrate preparation. Lane 1, uracil-containing 60-mer; lane 2, uracil-containing 60-mer, digested with uracil DNA glycosylase and tested with T4-pdg (control of AP-site formation); reduced AP site-containing DNA before (lane 3) and after (lanes 4–6) purification; DPC-containing DNA before (lane 7) and after (lanes 8–10) purification. After purification, DNAs were subjected to the restriction endonuclease digestion with Sna BI ( , ) or Hae III ( , ). ( C ) SDS/PAGE showing DPC-containing DNA substrates before (lane 1) and after (lane 2) Hae III digestion.

    Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Purification, SDS Page

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

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

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

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

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

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

    11) Product Images from "Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism"

    Article Title: Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006201

    Cleavage reactions with transposase mutants and an abasic substrate. Transpososomes were first assembled in the absence of divalent metal ions. The cleavage reaction was initiated by the addition of MgCl 2 at time zero. Aliquots were withdrawn at the indicated times and the reaction halted by the addition of EDTA and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography on a phosphoimager. The DNA substrates were labeled at both 5′-ends so that all three phosphoryl transfer reactions could be observed in a single experiment. The steps of the cleavage reaction are shown in panel A of the figure below the gel panel. The flanking DNA is to the left and the transposon arm to the right of the half bracket that indicates the location of the transposon end. The positions of the radioactive labels are indicated by the asterisks. Since the reactions are analyzed on denaturing gels, the unlabeled DNA strands, illustrated in grey, are not detected in the autoradiograms. The identity of each band is indicated to the right of the gel in panel A. Bands I and IV each represent a single product of the reaction as indicated. Bands II and III each represent mixtures of more than one co-migrating product and/or substrate as indicated. A B Cleavage reactions of wild type and abasic DNA substrates. The diagonal slashes indicate regions of the gels that have been removed because they contain no relevant information. Unaltered images of the gels are provided in Figure S1 . The identity of the products are indicated next to each band: Band I is the hairpin intermediate; Band II consists the unreacted substrate plus the top strand of the nicked product; Band III contains the bottom strand of the nicked product and the bottom strand of the cleaved transposon end (the resolved hairpin); Band IV contains the top strand of the cleaved flanking DNA that is released upon hairpin formation. In panel B the substrate has an abasic residue at position +2 of the top strand. This was prepared by incorporating a uracil residue at that position by PCR and subsequently treating the substrate with uracil glycosylase. This approach was preferred over one in which the abasic site could have been incorporated during oligonucleotide synthesis. Tn 10 transposon arms are folded during assembly of the transpososome [32] , [39] , [40] , and the DNA fragments required are too long for convenient oligonucleotide synthesis. C-F Quantification of cleavage intermediates. The respective products are plotted as a percentage of the total substrate in the reaction. The amount of each intermediate present at each time point is indicated by the shading within the column. None of the conditions tested severely inhibit the nicking step of the reaction. Sixty minutes is sufficient time for all of the transpososome complexes present at the start of the reaction to achieve the first nick. The height of the column at the 60 minute time point is therefore equivalent to the efficiency of transposome assembly, which varied over a 3-fold range in the reactions presented in this experiment. Bands I and IV (corresponding to the hairpin and cleaved top strand, respectively) are unique and unambiguous products of the reaction and can be quantified directly from the gel by phosphorimager analysis. Other intermediates and/or substrate comigrate and therefore can not be quantified directly. They were calculated as follows: first strand cleavage (first nick) = Band III - (Band IV - Band I). Hairpin resolution = Band IV - Band I. These calculations rely on equal labeling efficiency at either end of the substrate. To determine the efficiency of labeling an aliquot of the substrate was cleaved into two parts by NdeI, and the ratio of label incorporated at each end of the fragment was determined by phosphoimager analysis. This ratio was used to adjust all quantifications described above.
    Figure Legend Snippet: Cleavage reactions with transposase mutants and an abasic substrate. Transpososomes were first assembled in the absence of divalent metal ions. The cleavage reaction was initiated by the addition of MgCl 2 at time zero. Aliquots were withdrawn at the indicated times and the reaction halted by the addition of EDTA and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography on a phosphoimager. The DNA substrates were labeled at both 5′-ends so that all three phosphoryl transfer reactions could be observed in a single experiment. The steps of the cleavage reaction are shown in panel A of the figure below the gel panel. The flanking DNA is to the left and the transposon arm to the right of the half bracket that indicates the location of the transposon end. The positions of the radioactive labels are indicated by the asterisks. Since the reactions are analyzed on denaturing gels, the unlabeled DNA strands, illustrated in grey, are not detected in the autoradiograms. The identity of each band is indicated to the right of the gel in panel A. Bands I and IV each represent a single product of the reaction as indicated. Bands II and III each represent mixtures of more than one co-migrating product and/or substrate as indicated. A B Cleavage reactions of wild type and abasic DNA substrates. The diagonal slashes indicate regions of the gels that have been removed because they contain no relevant information. Unaltered images of the gels are provided in Figure S1 . The identity of the products are indicated next to each band: Band I is the hairpin intermediate; Band II consists the unreacted substrate plus the top strand of the nicked product; Band III contains the bottom strand of the nicked product and the bottom strand of the cleaved transposon end (the resolved hairpin); Band IV contains the top strand of the cleaved flanking DNA that is released upon hairpin formation. In panel B the substrate has an abasic residue at position +2 of the top strand. This was prepared by incorporating a uracil residue at that position by PCR and subsequently treating the substrate with uracil glycosylase. This approach was preferred over one in which the abasic site could have been incorporated during oligonucleotide synthesis. Tn 10 transposon arms are folded during assembly of the transpososome [32] , [39] , [40] , and the DNA fragments required are too long for convenient oligonucleotide synthesis. C-F Quantification of cleavage intermediates. The respective products are plotted as a percentage of the total substrate in the reaction. The amount of each intermediate present at each time point is indicated by the shading within the column. None of the conditions tested severely inhibit the nicking step of the reaction. Sixty minutes is sufficient time for all of the transpososome complexes present at the start of the reaction to achieve the first nick. The height of the column at the 60 minute time point is therefore equivalent to the efficiency of transposome assembly, which varied over a 3-fold range in the reactions presented in this experiment. Bands I and IV (corresponding to the hairpin and cleaved top strand, respectively) are unique and unambiguous products of the reaction and can be quantified directly from the gel by phosphorimager analysis. Other intermediates and/or substrate comigrate and therefore can not be quantified directly. They were calculated as follows: first strand cleavage (first nick) = Band III - (Band IV - Band I). Hairpin resolution = Band IV - Band I. These calculations rely on equal labeling efficiency at either end of the substrate. To determine the efficiency of labeling an aliquot of the substrate was cleaved into two parts by NdeI, and the ratio of label incorporated at each end of the fragment was determined by phosphoimager analysis. This ratio was used to adjust all quantifications described above.

    Techniques Used: DNA Sequencing, Autoradiography, Labeling, Polymerase Chain Reaction, Oligonucleotide Synthesis

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

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

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

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157860

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

    Techniques Used: Activity Assay, Negative Control

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

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

    14) Product Images from "Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroiminohydantoin and guanidinohydantoin"

    Article Title: Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroiminohydantoin and guanidinohydantoin

    Journal:

    doi: 10.1016/j.dnarep.2013.04.026

    3.4 Human NEIL3 acts mainly as a monofunctional DNA glycosylase with highest affinity for the hydantoin lesions Sp and Gh
    Figure Legend Snippet: 3.4 Human NEIL3 acts mainly as a monofunctional DNA glycosylase with highest affinity for the hydantoin lesions Sp and Gh

    Techniques Used:

    Residues involved in DNA glycosylase and AP lyase activity of human NEIL3
    Figure Legend Snippet: Residues involved in DNA glycosylase and AP lyase activity of human NEIL3

    Techniques Used: Activity Assay

    15) Product Images from "Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine"

    Article Title: Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine

    Journal:

    doi: 10.1016/j.dnarep.2012.06.004

    DNA glycosylase activity of AthFpgΔ88 WI187-188IY and EcoFpg IY170-171WI. Excess active enzyme (50 nM) was incubated with double-stranded substrate (10 nM) containing 8-oxoG, MeFapyG, Gh, or Sp1 opposite C. Each glycosylase reaction was incubated
    Figure Legend Snippet: DNA glycosylase activity of AthFpgΔ88 WI187-188IY and EcoFpg IY170-171WI. Excess active enzyme (50 nM) was incubated with double-stranded substrate (10 nM) containing 8-oxoG, MeFapyG, Gh, or Sp1 opposite C. Each glycosylase reaction was incubated

    Techniques Used: Activity Assay, Incubation

    Glycosylase/lyase activity assays and activity profile on γ-irradiated DNA of wild-type EcoFpg and EcoFpgΔ213-229. (A) The glycosylase assay was performed by incubating 10 nM of double-stranded substrate containing 8-oxoG:C, MeFapy:C,
    Figure Legend Snippet: Glycosylase/lyase activity assays and activity profile on γ-irradiated DNA of wild-type EcoFpg and EcoFpgΔ213-229. (A) The glycosylase assay was performed by incubating 10 nM of double-stranded substrate containing 8-oxoG:C, MeFapy:C,

    Techniques Used: Activity Assay, Irradiation

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    Amplification:

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    Binding Assay:

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    Construct:

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    Real-time Polymerase Chain Reaction:

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    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