uracil dna glycosylase (New England Biolabs)

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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Uracil DNA Glycosylase UDG 5 000 units
https://www.bioz.com/result/uracil dna glycosylase/product/New England Biolabs
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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

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

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. ... 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.](https://storage.googleapis.com/bioz_article_images/PMC2704287/carcinbgp118f04_ht.jpg)
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

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

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

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

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

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

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

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

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

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
![... and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography ... 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.](https://storage.googleapis.com/bioz_article_images/PMC2705183/pone.0006201.g004.jpg)
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

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

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 ... 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.](https://storage.googleapis.com/bioz_article_images/PMC4982613/pone.0157860.g001.jpg)
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

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:

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

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

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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Sequencing libraries were generated from each DNA extract following a single-stranded library preparation protocol [ ], which included treatment with 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 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 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 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 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 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 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). .. 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 HRM Assay:Article Title: Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors Article Snippet: For Plasmid Preparation:Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase Article Snippet: The supernatant was incubated with 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 |