e.coli uracil dna glycosylase Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs e coli uracil dna glycosylase
    <t>DNA</t> <t>glycosylase/lyase</t> 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),
    E Coli Uracil Dna Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli uracil dna glycosylase/product/New England Biolabs
    Average 99 stars, based on 123 article reviews
    Price from $9.99 to $1999.99
    e coli uracil dna glycosylase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    97
    Thermo Fisher e coli uracil dna glycosylase
    Effects of EDTA on MutY Dr binding and <t>glycosylase</t> activities with A/G- and A/GO-containing <t>DNA.</t> (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).
    E Coli Uracil Dna Glycosylase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli uracil dna glycosylase/product/Thermo Fisher
    Average 97 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    e coli uracil dna glycosylase - by Bioz Stars, 2020-07
    97/100 stars
      Buy from Supplier

    93
    Millipore e coli uracil dna glycosylase
    Effects of EDTA on MutY Dr binding and <t>glycosylase</t> activities with A/G- and A/GO-containing <t>DNA.</t> (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).
    E Coli Uracil Dna Glycosylase, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli uracil dna glycosylase/product/Millipore
    Average 93 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    e coli uracil dna glycosylase - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    88
    Trevigen e coli uracil dna glycosylase
    Effects of EDTA on MutY Dr binding and <t>glycosylase</t> activities with A/G- and A/GO-containing <t>DNA.</t> (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).
    E Coli Uracil Dna Glycosylase, supplied by Trevigen, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli uracil dna glycosylase/product/Trevigen
    Average 88 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    e coli uracil dna glycosylase - by Bioz Stars, 2020-07
    88/100 stars
      Buy from Supplier

    85
    Trevigen escherichia coli mismatch uracil dna glycosylase mug
    Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer <t>DNA</t> duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA <t>glycosylase</t> assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( <t>MUG</t> ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).
    Escherichia Coli Mismatch Uracil Dna Glycosylase Mug, supplied by Trevigen, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli mismatch uracil dna glycosylase mug/product/Trevigen
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    escherichia coli mismatch uracil dna glycosylase mug - by Bioz Stars, 2020-07
    85/100 stars
      Buy from Supplier

    93
    Roche uracil dna glycosylase
    Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer <t>DNA</t> duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA <t>glycosylase</t> assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( <t>MUG</t> ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).
    Uracil Dna Glycosylase, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/uracil dna glycosylase/product/Roche
    Average 93 stars, based on 424 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    86
    New England Biolabs e coli uracil dna glycosilase
    Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer <t>DNA</t> duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA <t>glycosylase</t> assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( <t>MUG</t> ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).
    E Coli Uracil Dna Glycosilase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli uracil dna glycosilase/product/New England Biolabs
    Average 86 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    e coli uracil dna glycosilase - by Bioz Stars, 2020-07
    86/100 stars
      Buy from Supplier

    85
    Roche escherichia coli uracil n glycosylase
    Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer <t>DNA</t> duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA <t>glycosylase</t> assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( <t>MUG</t> ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).
    Escherichia Coli Uracil N Glycosylase, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli uracil n glycosylase/product/Roche
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    escherichia coli uracil n glycosylase - by Bioz Stars, 2020-07
    85/100 stars
      Buy from Supplier

    95
    Thermo Fisher amperase uracil n glycosylase
    Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer <t>DNA</t> duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA <t>glycosylase</t> assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( <t>MUG</t> ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).
    Amperase Uracil N Glycosylase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amperase uracil n glycosylase/product/Thermo Fisher
    Average 95 stars, based on 481 article reviews
    Price from $9.99 to $1999.99
    amperase uracil n glycosylase - by Bioz Stars, 2020-07
    95/100 stars
      Buy from Supplier

    92
    Trevigen enzyme buffer
    Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer <t>DNA</t> duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA <t>glycosylase</t> assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( <t>MUG</t> ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).
    Enzyme Buffer, supplied by Trevigen, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/enzyme buffer/product/Trevigen
    Average 92 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    enzyme buffer - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    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

    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

    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

    Effects of EDTA on MutY Dr binding and glycosylase activities with A/G- and A/GO-containing DNA. (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).

    Journal: Journal of Bacteriology

    Article Title: Molecular Cloning and Functional Analysis of the MutY Homolog of Deinococcus radiodurans

    doi: 10.1128/JB.183.21.6151-6158.2001

    Figure Lengend Snippet: Effects of EDTA on MutY Dr binding and glycosylase activities with A/G- and A/GO-containing DNA. (A) Glycosylase activity. Oligonucleotide 20-mer DNA containing an A/G (lanes 1 to 7) or A/GO (lanes 8 to 14) mismatch was reacted with MutY Dr for 30 min at 37°C in the presence of different EDTA concentrations. MutY Dr reactions were carried out in buffers in the presence of 1 mM (lanes 1 and 8), 5 mM (lanes 2 and 9), 10 mM (lanes 3 and 10), 20 mM (lanes 4 and 11), 30 mM (lanes 5 and 12), 40 mM (lanes 6 and 13), and 50 mM (lanes 7 and 14) EDTA. The reaction products were analyzed on a 14% polyacrylamide DNA sequencing gel. The arrows indicate the positions of intact oligonucleotide (arrow I) and nicking product (arrow N). (B) DNA binding activity. The EDTA concentrations used were similar to those used in the experiment whose results are shown in panel A. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mM Tris borate (pH 8.3)–1 mM EDTA. The arrows indicate the positions of protein-bound DNA (arrow B), protein-free double-stranded DNA (arrow F), and single-stranded DNA (arrow S).

    Article Snippet: DNA substrates containing U/G or U/GO (300 fmol) were fully converted to AP/G- or AP/GO-containing DNA substrates by treatment with 1.5 U of E. coli uracil DNA glycosylase (Life Technologies, Inc.) at 37°C for 1 h in a buffer containing 20 mM Tris-HCl (pH 7.6), 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 2.9% glycerol.

    Techniques: Binding Assay, Activity Assay, DNA Sequencing

    Glycosylase activities of MutY Dr (DrMutY) and MutY Ec (EcMutY) with different mismatches. Oligonucleotide substrates (3′-end-labeled 20-mers; 1.8 fmol) containing the mismatches indicated above the lanes were incubated with 3.6 nM MutY Dr (lanes 1 to 8) or 3.6 nM MutY Ec (lanes 9 to 16) for 30 min at 37°C. Abbreviations: O, GO; 2, 2-aminopurine. The arrows indicate the positions of intact DNA substrate (arrow I) and the cleaved DNA fragment (arrow N). The minor band above the cleaved product was derived from an impurity of DNA substrates that appeared above the intact DNA.

    Journal: Journal of Bacteriology

    Article Title: Molecular Cloning and Functional Analysis of the MutY Homolog of Deinococcus radiodurans

    doi: 10.1128/JB.183.21.6151-6158.2001

    Figure Lengend Snippet: Glycosylase activities of MutY Dr (DrMutY) and MutY Ec (EcMutY) with different mismatches. Oligonucleotide substrates (3′-end-labeled 20-mers; 1.8 fmol) containing the mismatches indicated above the lanes were incubated with 3.6 nM MutY Dr (lanes 1 to 8) or 3.6 nM MutY Ec (lanes 9 to 16) for 30 min at 37°C. Abbreviations: O, GO; 2, 2-aminopurine. The arrows indicate the positions of intact DNA substrate (arrow I) and the cleaved DNA fragment (arrow N). The minor band above the cleaved product was derived from an impurity of DNA substrates that appeared above the intact DNA.

    Article Snippet: DNA substrates containing U/G or U/GO (300 fmol) were fully converted to AP/G- or AP/GO-containing DNA substrates by treatment with 1.5 U of E. coli uracil DNA glycosylase (Life Technologies, Inc.) at 37°C for 1 h in a buffer containing 20 mM Tris-HCl (pH 7.6), 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 2.9% glycerol.

    Techniques: Labeling, Incubation, Derivative Assay

    Endo VIII activity on the DNA products generated by MutY, MutY-(1–226) and UDG

    Journal: Biochemical Journal

    Article Title: Physical and functional interactions between Escherichia coli MutY and endonuclease VIII

    doi: 10.1042/BJ20051133

    Figure Lengend Snippet: Endo VIII activity on the DNA products generated by MutY, MutY-(1–226) and UDG

    Article Snippet: E. coli UDG (uracil DNA glycosylase) and Endo VIII were purchase from Invitrogen and New England Biolabs respectively.

    Techniques: Activity Assay, Generated

    Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer DNA duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA glycosylase assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( MUG ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).

    Journal: The Journal of Biological Chemistry

    Article Title: Structural Basis for the Inhibition of Human Alkyladenine DNA Glycosylase (AAG) by 3,N4-Ethenocytosine-containing DNA *

    doi: 10.1074/jbc.M110.192435

    Figure Lengend Snippet: Biochemical characterization of AAG variants with oligomers containing etheno lesions. A , representative gels showing the results of gel mobility shift assays for Δ79AAG binding to ϵA:T (ϵA paired opposite T) and ϵC:G (ϵC paired opposite G) 25-mer DNA duplexes. 2 n m 32 P-labeled oligonucleotide was incubated with an indicated concentration of Δ79AAG in the 1× binding buffer, and the resulting protein-DNA complexes were resolved using 6% native-PAGE. The top band corresponds to the protein-DNA complex, and the bottom band corresponds to the free DNA. B , graphical representation of Δ79AAG binding to ϵA:T 25-mer (■), ϵC:G 25-mer (□), ϵA:T 13-mer (●), and ϵC:G 13-mer (○) oligonucleotide duplexes as measured by gel shift mobility assays. Error bars indicate the S.D. of at least three independent trials. C , representative gels showing the results from competition DNA glycosylase assays. The activity of Δ79AAG on labeled ϵA:T 25-mer duplex is inhibited in the presence of indicated concentrations of ϵA:T 13-mer and ϵC:G 13-mer unlabeled competitors. 2 n m 32 P-labeled ϵA:T 25-mer duplex oligonucleotide was incubated in 1× glycosylase assay buffer with 5 n m Δ79AAG enzyme and increasing concentrations of cold competitors at 37 °C for 30 min. Following hot alkali treatment, the products were resolved using 20% denaturing urea-PAGE. The top band represents the uncleaved DNA substrate, and the bottom band represents the cleaved product. D , graphical representation of the data from competition DNA glycosylase assays, looking at the inhibition of Δ79AAG activity on labeled ϵA:T 25-mer duplex substrate by ϵA:T 13-mer (●) and ϵC:G 13-mer (○) unlabeled competitors. Error bars indicate the S.D. of at least three independent trials. E , gel results of DNA glycosylase assays for full-length AAG ( FL-AAG ) and truncated Δ79AAG on ϵA:T and ϵC:G 25-mer DNA duplexes with E. coli mismatch uracil DNA glycosylase ( MUG ) as a positive control. Single strand breaks are observed in this urea-denaturing gel when piperidine treatment cleaves AP sites ( bottom band ). When AAG fails to cleave the lesion base, no AP site is formed, and the oligomer remains intact ( top band ).

    Article Snippet: In contrast, the positive control, Escherichia coli mismatch uracil DNA glycosylase (MUG) (Trevigen, Inc.), shows robust catalytic activity on ϵC contained in an ϵC:G 25-mer duplex ( E ).

    Techniques: Mobility Shift, Binding Assay, Labeling, Incubation, Concentration Assay, Clear Native PAGE, Electrophoretic Mobility Shift Assay, Activity Assay, Polyacrylamide Gel Electrophoresis, Inhibition, Positive Control