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    New England Biolabs glycosylases uracil dna glycosylase ung
    Ability of <t>UNG</t> and Arg 276 mutant proteins to form UV-catalyzed cross-links to [ 32 P]25-mer <t>DNA</t> A , samples (12 µl) containing 20 pmol of 5′-end 32 P-labeled oligonucleotide T-25-mer, U-25-mer, or dψU-25-mer without UNG ( lanes 1–3 , respectively) or with 40 pmol of UNG ( lanes 4–6 , respectively) were UV-irradiated for 30 min as described under “Experimental Procedures.” Following irradiation, samples were subjected to non-denaturing polyacrylamide gel electrophoresis; the gels were dried and analyzed using a PhosphorImager. The positions of the UNG × [ 32 P]DNA-25-mer cross-linked complex bands and free [ 32 P]DNA-25-mer bands are indicated by arrows . B , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min ( lanes 3–14 , respectively), reactions were analyzed as in A . Control reactions ( lanes 1 and 2 ), containing 20 pmol of [ 32 P]dψU-25-mer, were not irradiated. C , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer ( closed circles ), [ 32 P]T-25-mer ( closed squares ), or [ 32 P]U-25-mer ( closed triangles ), and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min, reactions were analyzed as in A , and the PhosphorImager data were quantified using the ImageQuant program. The cross-linking efficiency (%) was calculated by dividing the intensity of the UNG × [ 32 P]25-mer band by the sum of the [ 32 P]25-mer and UNG × [ 32 P]25-mer bands and multiplying by 100. D , reaction mixtures (12 µl) were prepared that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of each Arg 276 mutant enzyme. The reactions were UV-irradiated for 10 min and analyzed as described in C . The cross-linking efficiency of each mutant preparation, indicated by the corresponding single letter amino acid abbreviation, is compared with that of UNG. Error bars represent the standard deviation of three experiments.
    Glycosylases Uracil Dna Glycosylase Ung, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs uracil dna glycosylase inhibitor
    Facilitated site transfer by human uracil <t>DNA</t> <t>glycosylase</t> (hUNG). In all facilitated transfer assays the hUNG concentration is 5–20 pM and the DNA substrate concentration is 40 nM. (a) Facilitated site transfer of hUNG between two uracil sites on the same DNA strand separated by 10 bps (S10). Reactions in the absence and presence of 10 mM uracil are shown. Facilitated transfer is qualitatively indicated by an excess of double excision fragments (A and C) relative single site excision products (AB and BC). (b) Facilitated site transfer by hUNG between sites separated by 5 bp (S5) in the absence and presence of 10 mM uracil. (c) Observed probability of site transfer (P trans obs ) as a function of time and uracil concentration for the substrate with a 10 bp site separation. Linear extrapolation to the y axis provides the true transfer probability at zero time (P trans ). (d) P trans ′ as a function of increasing uracil for substrates with 5, 10 and 20 bp site spacings. Each data point represents an individual experiment as in panels a and b and the prime notation indicates correction for the efficiency of excision (see text). The non-linear least squares fits in (d) use a kinetic partitioning model that relates the dependence of the total transfer probability (P trans ′ = P slide ′ + P hop ′) to the uracil trap concentration ( Supplementary Methods ). Uncut gel images are shown in Supplementary Fig. 6 . Error bars represent the mean ± 1 s.d. of at least three independent trials.
    Uracil Dna Glycosylase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ability of UNG and Arg 276 mutant proteins to form UV-catalyzed cross-links to [ 32 P]25-mer DNA A , samples (12 µl) containing 20 pmol of 5′-end 32 P-labeled oligonucleotide T-25-mer, U-25-mer, or dψU-25-mer without UNG ( lanes 1–3 , respectively) or with 40 pmol of UNG ( lanes 4–6 , respectively) were UV-irradiated for 30 min as described under “Experimental Procedures.” Following irradiation, samples were subjected to non-denaturing polyacrylamide gel electrophoresis; the gels were dried and analyzed using a PhosphorImager. The positions of the UNG × [ 32 P]DNA-25-mer cross-linked complex bands and free [ 32 P]DNA-25-mer bands are indicated by arrows . B , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min ( lanes 3–14 , respectively), reactions were analyzed as in A . Control reactions ( lanes 1 and 2 ), containing 20 pmol of [ 32 P]dψU-25-mer, were not irradiated. C , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer ( closed circles ), [ 32 P]T-25-mer ( closed squares ), or [ 32 P]U-25-mer ( closed triangles ), and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min, reactions were analyzed as in A , and the PhosphorImager data were quantified using the ImageQuant program. The cross-linking efficiency (%) was calculated by dividing the intensity of the UNG × [ 32 P]25-mer band by the sum of the [ 32 P]25-mer and UNG × [ 32 P]25-mer bands and multiplying by 100. D , reaction mixtures (12 µl) were prepared that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of each Arg 276 mutant enzyme. The reactions were UV-irradiated for 10 min and analyzed as described in C . The cross-linking efficiency of each mutant preparation, indicated by the corresponding single letter amino acid abbreviation, is compared with that of UNG. Error bars represent the standard deviation of three experiments.

    Journal: The Journal of biological chemistry

    Article Title: Mutational Analysis of Arginine 276 in the Leucine-loop of Human Uracil-DNA Glycosylase *

    doi: 10.1074/jbc.M407836200

    Figure Lengend Snippet: Ability of UNG and Arg 276 mutant proteins to form UV-catalyzed cross-links to [ 32 P]25-mer DNA A , samples (12 µl) containing 20 pmol of 5′-end 32 P-labeled oligonucleotide T-25-mer, U-25-mer, or dψU-25-mer without UNG ( lanes 1–3 , respectively) or with 40 pmol of UNG ( lanes 4–6 , respectively) were UV-irradiated for 30 min as described under “Experimental Procedures.” Following irradiation, samples were subjected to non-denaturing polyacrylamide gel electrophoresis; the gels were dried and analyzed using a PhosphorImager. The positions of the UNG × [ 32 P]DNA-25-mer cross-linked complex bands and free [ 32 P]DNA-25-mer bands are indicated by arrows . B , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min ( lanes 3–14 , respectively), reactions were analyzed as in A . Control reactions ( lanes 1 and 2 ), containing 20 pmol of [ 32 P]dψU-25-mer, were not irradiated. C , reaction mixtures (12 µl) were prepared in duplicate that contained 20 pmol of [ 32 P]dψU-25-mer ( closed circles ), [ 32 P]T-25-mer ( closed squares ), or [ 32 P]U-25-mer ( closed triangles ), and 40 pmol of UNG. Following UV irradiation for 0, 5, 10, 20, 30, and 45 min, reactions were analyzed as in A , and the PhosphorImager data were quantified using the ImageQuant program. The cross-linking efficiency (%) was calculated by dividing the intensity of the UNG × [ 32 P]25-mer band by the sum of the [ 32 P]25-mer and UNG × [ 32 P]25-mer bands and multiplying by 100. D , reaction mixtures (12 µl) were prepared that contained 20 pmol of [ 32 P]dψU-25-mer and 40 pmol of each Arg 276 mutant enzyme. The reactions were UV-irradiated for 10 min and analyzed as described in C . The cross-linking efficiency of each mutant preparation, indicated by the corresponding single letter amino acid abbreviation, is compared with that of UNG. Error bars represent the standard deviation of three experiments.

    Article Snippet: The nucleotide sequence corresponding to the core catalytic domain of human uracil-DNA glycosylase (UNG*) was amplified in a polymerase chain reaction (100 µl) containing pUNG15 as template (0.5 µg), primers (1 µ m each) RI: 5′-GC GAATTC TTTGGAGAGAGCTGGAAG-3′, and H3: 5′-GC AAGCTT TCACAGCTCCTTCCAGTC-3′, as forward and reverse primers, respectively, 1× ThermoPol (New England Biolabs) buffer, 200 µ m each dATP, dTTP, dCTP, and dGTP, and 2 units of Deep Vent DNA polymerase.

    Techniques: Mutagenesis, Labeling, Irradiation, Polyacrylamide Gel Electrophoresis, Standard Deviation

    Tertiary structure of human uracil-DNA glycosylase bound to DNA ). The DNA is shown in yellow and the view is looking into the major groove. A , three distinct amino acid sequences ( red tubes ) of the UNG* polypeptide backbone ( silver tubes ) are shown. The 4 Pro-Ser loop ( 165 PPPPS 169 ) and the Gly-Ser loop ( 246 GS 247 ). The Leu 272 loop ( 268 HPSPLSVYR 276 ), which contains Arg 276 ). Conserved amino acid residues (Gln 144 , Asn 204 , and His 268 ) in the UNG* binding pocket capture (“pull”) and stabilize the expelled extrahelical dψU. α-Helices are depicted as silver cylinders and β-sheets are illustrated as blue strands . B , ball-and-stick diagram of the UNG Leu 272 loop ( 271 PLSVYR 276 ) shown in silver and a portion of the oligonucleotide sequence 3′-CTA dψU-5′ shown in yellow . The ηN of the Arg 276 guanidinium side chain (nitrogen atoms, blue balls ) is shown as interacting ( black rippled lines ) with the 5′-phosphate of the cytosine residue (oxygen atom, red ball ), as stated for cleaved U·G DNA by Slupphaug et al. ). The εN participates in water-bridged (water, black ball ) hydrogen bonding ( dashed lines ) with the N3 of adenine (blue ball) and the carbonyl group ( red ball ) of Leu 272 as shown in Parikh et al. ). Structures were drawn with the Cn3D 4.0 software program using Protein Data Bank code 1EMH (MMDB 13471) deposited by Parikh et al. ) in the Molecular Modeling Data base of the National Center for Biotechnology Information.

    Journal: The Journal of biological chemistry

    Article Title: Mutational Analysis of Arginine 276 in the Leucine-loop of Human Uracil-DNA Glycosylase *

    doi: 10.1074/jbc.M407836200

    Figure Lengend Snippet: Tertiary structure of human uracil-DNA glycosylase bound to DNA ). The DNA is shown in yellow and the view is looking into the major groove. A , three distinct amino acid sequences ( red tubes ) of the UNG* polypeptide backbone ( silver tubes ) are shown. The 4 Pro-Ser loop ( 165 PPPPS 169 ) and the Gly-Ser loop ( 246 GS 247 ). The Leu 272 loop ( 268 HPSPLSVYR 276 ), which contains Arg 276 ). Conserved amino acid residues (Gln 144 , Asn 204 , and His 268 ) in the UNG* binding pocket capture (“pull”) and stabilize the expelled extrahelical dψU. α-Helices are depicted as silver cylinders and β-sheets are illustrated as blue strands . B , ball-and-stick diagram of the UNG Leu 272 loop ( 271 PLSVYR 276 ) shown in silver and a portion of the oligonucleotide sequence 3′-CTA dψU-5′ shown in yellow . The ηN of the Arg 276 guanidinium side chain (nitrogen atoms, blue balls ) is shown as interacting ( black rippled lines ) with the 5′-phosphate of the cytosine residue (oxygen atom, red ball ), as stated for cleaved U·G DNA by Slupphaug et al. ). The εN participates in water-bridged (water, black ball ) hydrogen bonding ( dashed lines ) with the N3 of adenine (blue ball) and the carbonyl group ( red ball ) of Leu 272 as shown in Parikh et al. ). Structures were drawn with the Cn3D 4.0 software program using Protein Data Bank code 1EMH (MMDB 13471) deposited by Parikh et al. ) in the Molecular Modeling Data base of the National Center for Biotechnology Information.

    Article Snippet: The nucleotide sequence corresponding to the core catalytic domain of human uracil-DNA glycosylase (UNG*) was amplified in a polymerase chain reaction (100 µl) containing pUNG15 as template (0.5 µg), primers (1 µ m each) RI: 5′-GC GAATTC TTTGGAGAGAGCTGGAAG-3′, and H3: 5′-GC AAGCTT TCACAGCTCCTTCCAGTC-3′, as forward and reverse primers, respectively, 1× ThermoPol (New England Biolabs) buffer, 200 µ m each dATP, dTTP, dCTP, and dGTP, and 2 units of Deep Vent DNA polymerase.

    Techniques: Binding Assay, Sequencing, Software

    Specific uracil-DNA glycosylase activity of R276X mutant proteins ) and the Protein Assay reagent (Bio-Rad). The specific activity (units/mg) of UNG* and the R276X mutant enzymes was normalized to that of UNG (3.76 × 10 5 units/mg), which was defined as 100%. Arg 276 mutant proteins are denoted by single letter amino acid abbreviations. Error bars represent the standard deviation of four experimental determinations.

    Journal: The Journal of biological chemistry

    Article Title: Mutational Analysis of Arginine 276 in the Leucine-loop of Human Uracil-DNA Glycosylase *

    doi: 10.1074/jbc.M407836200

    Figure Lengend Snippet: Specific uracil-DNA glycosylase activity of R276X mutant proteins ) and the Protein Assay reagent (Bio-Rad). The specific activity (units/mg) of UNG* and the R276X mutant enzymes was normalized to that of UNG (3.76 × 10 5 units/mg), which was defined as 100%. Arg 276 mutant proteins are denoted by single letter amino acid abbreviations. Error bars represent the standard deviation of four experimental determinations.

    Article Snippet: The nucleotide sequence corresponding to the core catalytic domain of human uracil-DNA glycosylase (UNG*) was amplified in a polymerase chain reaction (100 µl) containing pUNG15 as template (0.5 µg), primers (1 µ m each) RI: 5′-GC GAATTC TTTGGAGAGAGCTGGAAG-3′, and H3: 5′-GC AAGCTT TCACAGCTCCTTCCAGTC-3′, as forward and reverse primers, respectively, 1× ThermoPol (New England Biolabs) buffer, 200 µ m each dATP, dTTP, dCTP, and dGTP, and 2 units of Deep Vent DNA polymerase.

    Techniques: Activity Assay, Mutagenesis, Standard Deviation

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.0030135

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

    Article Snippet: To each well was added 10 μl of cell lysate in NP40 buffer and 70 μl of a master mix containing 10 pmol Taqman probe, 0.4 units uracil DNA glycosylase (NEB, http://www.neb.com/ ), 50 mM Tris (pH 7.4), and 10 mM EDTA.

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm1053

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

    Article Snippet: Uracil DNA [Glycosylase (UDG) (M0280S), human AP endonuclease I (APE1) (M0282S), terminal transferase (M0252S), T4 PNK (M0201S)] and T4 DNA ligase (M0202S) were purchased from New England Biolabs.

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

    DNA glycosylase/lyase activity of MmuNeil3Δ324 and NEIL3Δ324. Single- and double-stranded substrates containing Tg, Sp, or an AP site (25 nM) were incubated with 25 nM active MmuNeil3Δ324 (lane 4) and NEIL3Δ324 (lane 5),

    Journal: Protein Expression and Purification

    Article Title: Expression and purification of active mouse and human NEIL3 proteins

    doi: 10.1016/j.pep.2012.04.022

    Figure Lengend Snippet: DNA glycosylase/lyase activity of MmuNeil3Δ324 and NEIL3Δ324. Single- and double-stranded substrates containing Tg, Sp, or an AP site (25 nM) were incubated with 25 nM active MmuNeil3Δ324 (lane 4) and NEIL3Δ324 (lane 5),

    Article Snippet: To create the substrate with an apurinic or apyrimidinic (AP) site, duplex or single-stranded oligodeoxynucleotides containing U were treated with E. coli uracil DNA glycosylase (New England Biolabs) at room temperature for 15 min.

    Techniques: Activity Assay, Incubation

    Uracil removal is performed by uracil DNA glycosylase, as shown by complete sensitivity to the UDG inhibitor Ugi

    Journal: Biochemistry

    Article Title: Base Excision Repair in Early Zebrafish Development: Evidence for DNA Polymerase Switching and Standby AP endonuclease Activity

    doi: 10.1021/bi900253d

    Figure Lengend Snippet: Uracil removal is performed by uracil DNA glycosylase, as shown by complete sensitivity to the UDG inhibitor Ugi

    Article Snippet: When uracil DNA glycosylase activity (Reaction 1) alone was measured, Substrate I, end-labeled at the 5′ end by means of polynucleotide kinase (New England Biolabs, Beverly MA) and [γ32 P]ATP, was used, Mg2+ was replaced with 4 mM EDTA, and the reaction was stopped by phenol extraction.

    Techniques:

    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

    Fluorescence assay of uracil DNA glycosylase activity. RK13 cells were mock infected or infected with recombinant virus at 10 PFU/cell. After 8 h at 37°C, the cells were harvested and lysed. Fluorescence assays were performed using 5 μg of cell extract protein and 1 μg of supercoiled pUC-19 DNA containing (A and B) or lacking (C and D) uracil residues. At the indicated times, aliquots of the reaction mixture were removed into pH 12 buffer, and fluorescence was measured before and after the mixtures were boiled. The percent fluorescence represents the amount of supercoiled pUC-19 remaining. The presence of nicks in the uracil containing pUC 19, prepared from E. coli CJ236 ( dut ung ) cells, accounted for the values of

    Journal: Journal of Virology

    Article Title: Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells

    doi: 10.1128/JVI.77.1.159-166.2003

    Figure Lengend Snippet: Fluorescence assay of uracil DNA glycosylase activity. RK13 cells were mock infected or infected with recombinant virus at 10 PFU/cell. After 8 h at 37°C, the cells were harvested and lysed. Fluorescence assays were performed using 5 μg of cell extract protein and 1 μg of supercoiled pUC-19 DNA containing (A and B) or lacking (C and D) uracil residues. At the indicated times, aliquots of the reaction mixture were removed into pH 12 buffer, and fluorescence was measured before and after the mixtures were boiled. The percent fluorescence represents the amount of supercoiled pUC-19 remaining. The presence of nicks in the uracil containing pUC 19, prepared from E. coli CJ236 ( dut ung ) cells, accounted for the values of

    Article Snippet: Assays with the uracil-DNA glycosylase inhibitor protein (UGI) (New England Biolabs) were performed as described above except that 10 U of UGI were incubated with protein from each lysate in reaction buffer for 10 min at room temperature followed by 20 min on ice prior to the addition of substrate DNA.

    Techniques: Fluorescence, Activity Assay, Infection, Recombinant

    Sequence alignment of uracil DNA glycosylase proteins of viruses and cells. The shaded segments indicate identical amino acids. The circles indicate conserved active-site residues. The solid circles indicate the conserved amino acids of the vaccinia virus uracil DNA glycosylase that were mutated in this study. The numbers of the amino acid residues of each protein are indicated at the left and right of the lines. HSV-1, herpes simplex virus type 1; VAC, vaccinia virus; MPXV, monkeypox virus; VAR, variola major virus; EVM, ectromelia virus; CMPV, camelpox virus; CPXV, cowpox virus; YAB, Yaba-like disease virus; SWPV, swinepox virus; MYX, myxoma virus; RFB, rabbit fibroma virus; LSD, lumpy skin disease virus; SPPV, sheeppox virus; MCV, molluscum contagiosum virus; FPV, fowlpox virus.

    Journal: Journal of Virology

    Article Title: Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells

    doi: 10.1128/JVI.77.1.159-166.2003

    Figure Lengend Snippet: Sequence alignment of uracil DNA glycosylase proteins of viruses and cells. The shaded segments indicate identical amino acids. The circles indicate conserved active-site residues. The solid circles indicate the conserved amino acids of the vaccinia virus uracil DNA glycosylase that were mutated in this study. The numbers of the amino acid residues of each protein are indicated at the left and right of the lines. HSV-1, herpes simplex virus type 1; VAC, vaccinia virus; MPXV, monkeypox virus; VAR, variola major virus; EVM, ectromelia virus; CMPV, camelpox virus; CPXV, cowpox virus; YAB, Yaba-like disease virus; SWPV, swinepox virus; MYX, myxoma virus; RFB, rabbit fibroma virus; LSD, lumpy skin disease virus; SPPV, sheeppox virus; MCV, molluscum contagiosum virus; FPV, fowlpox virus.

    Article Snippet: Assays with the uracil-DNA glycosylase inhibitor protein (UGI) (New England Biolabs) were performed as described above except that 10 U of UGI were incubated with protein from each lysate in reaction buffer for 10 min at room temperature followed by 20 min on ice prior to the addition of substrate DNA.

    Techniques: Sequencing

    Gene fragmentation. ( A ) Schematic of the CDH gene fragmentation process. PCR with TTP/dUTP mixtures is used to generate copies of the target gene in which uracil is randomly incorporated in place of thymine. The uracil-doped amplified DNA is subjected to a modified base-excision cascade in which uracil-DNA glycosylase excises the uracil bases generating abasic sites, which are cleaved by endonuclease IV, giving a single-strand nick that is converted to a double-strand break and blunt-ended by S1 nuclease. As the reaction cascade is initiated only at uracils, whose distribution along the sequence and among the PCR reaction products is random, the cascade generates a random and unbiased library of gene fragments, whose size distribution is solely dictated by the TTP/dUTP ratio. ( B ) dUTP-dose dependent fragmentation. SYBR-Safe stained 1% agarose gel of an ∼2.2-kb human p85α PCR-amplified cDNA ( right- hand lane), alongside the products of CDH fragmentation reactions using increasing amounts of dUTP (as percent of total TTP+dUTP concentration). The progressive decrease in modal size of the DNA distribution with increasing dUTP concentration is clearly seen.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

    doi: 10.1110/ps.062082606

    Figure Lengend Snippet: Gene fragmentation. ( A ) Schematic of the CDH gene fragmentation process. PCR with TTP/dUTP mixtures is used to generate copies of the target gene in which uracil is randomly incorporated in place of thymine. The uracil-doped amplified DNA is subjected to a modified base-excision cascade in which uracil-DNA glycosylase excises the uracil bases generating abasic sites, which are cleaved by endonuclease IV, giving a single-strand nick that is converted to a double-strand break and blunt-ended by S1 nuclease. As the reaction cascade is initiated only at uracils, whose distribution along the sequence and among the PCR reaction products is random, the cascade generates a random and unbiased library of gene fragments, whose size distribution is solely dictated by the TTP/dUTP ratio. ( B ) dUTP-dose dependent fragmentation. SYBR-Safe stained 1% agarose gel of an ∼2.2-kb human p85α PCR-amplified cDNA ( right- hand lane), alongside the products of CDH fragmentation reactions using increasing amounts of dUTP (as percent of total TTP+dUTP concentration). The progressive decrease in modal size of the DNA distribution with increasing dUTP concentration is clearly seen.

    Article Snippet: Amplified DNA was agarose gel-purified, spectrophotometrically quantitated, and incubated in restriction buffer 3 (NEB) with a cocktail of E. coli uracil-DNA glycosylase (UDG–NEB), E. coli endonuclease IV, S1-nuclease (Invitrogen), and calf intestinal phosphatase (NEB) at 37°C for 16 h. The resultant DNA fragment pool was purified using the Min-Elute kit (QIAGEN), size-selected by excision from agarose gels, and requantitated prior to capture in pCR-Blunt-II TOPO (Invitrogen).

    Techniques: Polymerase Chain Reaction, Amplification, Modification, Sequencing, Staining, Agarose Gel Electrophoresis, Concentration Assay

    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) Udg activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.

    Journal: Nucleic Acids Research

    Article Title: AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe

    doi: 10.1093/nar/gkr933

    Figure Lengend Snippet: Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) Udg activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.

    Article Snippet: To generate intact AP substrate or a nicked AP substrate with a 3′-dRP terminus, the uracil substrate was pretreated with uracil DNA glycosylase (Udg, NEB) or Udg and Nth (NEB), respectively, for 15 min at 37°C.

    Techniques: Activity Assay, Positive Control, Incubation, Sequencing

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

    Journal: Nature Chemical Biology

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

    doi: 10.1038/nchembio.764

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

    Article Snippet: At each time point 4 μl of the reaction mix was taken out and quenched with Uracil DNA Glycosylase Inhibitor (UGI) at a final concentration of 0.1 Units (New England Biolabs) or 50 nM final concentration of a highly potent duplex DNA inhibitor (2′-fluoro-2′-deoxyuridine paired with 4-methylindole in duplex DNA) , , both of which rapidly and efficiently quenched hUNG activity.

    Techniques: Concentration Assay