Structured Review

Boehringer Mannheim exonuclease iii
Enhancement of <t>UDGase</t> activity by exonuclease <t>III.</t> Decrease in fluorescence observed in the presence of different concentrations of exonuclease III. Open squares, UDGase; closed squares, UDGase + 40 U; triangles, UDGase +80 U; open circles, UDGase +175 U; closed circles, LmAP.
Exonuclease Iii, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/exonuclease iii/product/Boehringer Mannheim
Average 92 stars, based on 4 article reviews
Price from $9.99 to $1999.99
exonuclease iii - by Bioz Stars, 2020-11
92/100 stars

Images

1) Product Images from "Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease"

Article Title: Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

Journal: Nucleic Acids Research

doi:

Enhancement of UDGase activity by exonuclease III. Decrease in fluorescence observed in the presence of different concentrations of exonuclease III. Open squares, UDGase; closed squares, UDGase + 40 U; triangles, UDGase +80 U; open circles, UDGase +175 U; closed circles, LmAP.
Figure Legend Snippet: Enhancement of UDGase activity by exonuclease III. Decrease in fluorescence observed in the presence of different concentrations of exonuclease III. Open squares, UDGase; closed squares, UDGase + 40 U; triangles, UDGase +80 U; open circles, UDGase +175 U; closed circles, LmAP.

Techniques Used: Activity Assay, Fluorescence

2) Product Images from "Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates"

Article Title: Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Diagram of experimental design and intrinsic terminator RNA secondary structures. ( A ) Transcription was initiated at the promoter (bent rightward arrow) and the ternary complexes were walked proximal to the termination site (T) on double-stranded PCR-generated templates that were biotinylated (shaded circle containing the letter B) at one of the 5′ ends. Next, the stalled elongation complexes (shaded ellipse) were digested with Exo III (shaded circles, missing a piece) and then all four NTPs were added, allowing the stalled elongation complex to resume transcript elongation. 5′ and 3′ ends of the DNA are as indicated. ( B ) The RNA secondary structures of the three intrinsic terminators, λ tR2, T 7 Te, and rrn BT1, are shown. The underlined bases indicate the positions of transcript release and the length of the RNA product [λ tR2 and rrn , T. Kerppola and M.J.C., unpublished results) and T 7 )]. The RNA secondary structures are drawn to conform to the classical model of transcript termination, with a G+C-rich stem and U-rich region immediately downstream. It should be noted, however, that for the λ tR2 and the rrn BT1 terminators, the hairpin can be extended to include upstream adenosines and downstream uridines.
Figure Legend Snippet: Diagram of experimental design and intrinsic terminator RNA secondary structures. ( A ) Transcription was initiated at the promoter (bent rightward arrow) and the ternary complexes were walked proximal to the termination site (T) on double-stranded PCR-generated templates that were biotinylated (shaded circle containing the letter B) at one of the 5′ ends. Next, the stalled elongation complexes (shaded ellipse) were digested with Exo III (shaded circles, missing a piece) and then all four NTPs were added, allowing the stalled elongation complex to resume transcript elongation. 5′ and 3′ ends of the DNA are as indicated. ( B ) The RNA secondary structures of the three intrinsic terminators, λ tR2, T 7 Te, and rrn BT1, are shown. The underlined bases indicate the positions of transcript release and the length of the RNA product [λ tR2 and rrn , T. Kerppola and M.J.C., unpublished results) and T 7 )]. The RNA secondary structures are drawn to conform to the classical model of transcript termination, with a G+C-rich stem and U-rich region immediately downstream. It should be noted, however, that for the λ tR2 and the rrn BT1 terminators, the hairpin can be extended to include upstream adenosines and downstream uridines.

Techniques Used: Polymerase Chain Reaction, Generated

Assaying for intrinsic termination at the λ tR2 terminator on both ss- and dsDNA templates. ( A ) Stalled C 47 complexes (lanes 1 and 5) in TGK-B 40 M 1 were chased with 500 μM ATP, 500 μM GTP, 500 μM CTP, and 500 μM UTP for 10 min at 42°C in the presence of rifampicin at 20 μg/ml (lanes 2–4 and 6–8). Lanes 2 and 6 are aliquots of the chase reactions before the pellet was separated from the supernatant fluid, lanes 3 and 7 are the RNAs associated with the Dynabeads (P), and lanes 4 and 8 are the RNAs released into the supernatant fluid (S). The λ tR2 terminator is at positions +104 to +105 relative to the transcription start site, whereas the end of the DNA template or run-off occurs at position +177. Lanes: 1–4, no Exo III; 5–8, digested with Exo III (2,000 units/ml) for 5 min at 30°C. Quantitation of the total counts within a lane between the C 47 ). ( B ) Exo III-digested DNA was analyzed on a 6% denaturing PAGE gel and detected by chemiluminescence. The nontemplate strand was end-labeled with on the 5′ end with a biotin moiety. Lanes: 1, no Exo III; 2, Exo III at 2,000 units/ml for 5 min at 30°C. The initial DNA template, 315 nucleotides long was reduced to approximately 204 nucleotides after Exo III digestion.
Figure Legend Snippet: Assaying for intrinsic termination at the λ tR2 terminator on both ss- and dsDNA templates. ( A ) Stalled C 47 complexes (lanes 1 and 5) in TGK-B 40 M 1 were chased with 500 μM ATP, 500 μM GTP, 500 μM CTP, and 500 μM UTP for 10 min at 42°C in the presence of rifampicin at 20 μg/ml (lanes 2–4 and 6–8). Lanes 2 and 6 are aliquots of the chase reactions before the pellet was separated from the supernatant fluid, lanes 3 and 7 are the RNAs associated with the Dynabeads (P), and lanes 4 and 8 are the RNAs released into the supernatant fluid (S). The λ tR2 terminator is at positions +104 to +105 relative to the transcription start site, whereas the end of the DNA template or run-off occurs at position +177. Lanes: 1–4, no Exo III; 5–8, digested with Exo III (2,000 units/ml) for 5 min at 30°C. Quantitation of the total counts within a lane between the C 47 ). ( B ) Exo III-digested DNA was analyzed on a 6% denaturing PAGE gel and detected by chemiluminescence. The nontemplate strand was end-labeled with on the 5′ end with a biotin moiety. Lanes: 1, no Exo III; 2, Exo III at 2,000 units/ml for 5 min at 30°C. The initial DNA template, 315 nucleotides long was reduced to approximately 204 nucleotides after Exo III digestion.

Techniques Used: Quantitation Assay, Polyacrylamide Gel Electrophoresis, Labeling

3) Product Images from "Different Regulation of the p53 Core Domain Activities 3?-to-5? Exonuclease and Sequence-Specific DNA Binding"

Article Title: Different Regulation of the p53 Core Domain Activities 3?-to-5? Exonuclease and Sequence-Specific DNA Binding

Journal: Molecular and Cellular Biology

doi:

Protease treatment of p53 activates its exonucleolytic activity. (A and B) Wild-type p53 (A) or exonuclease III (Ex. III) (B) (150 ng) was digested with various amounts of thermolysin (40 U/mg), ranging from 4 ng to 20 pg. Exonuclease activity was measured after 15 min of digestion. The amount of exonuclease III was chosen to match the exonuclease activity of 150 ng of wt p53. Incubation of p53 with buffer alone for 15 min (p53 buffer control) did not affect the p53 exonuclease activity. (C) Western blot analysis of p53 fragments resulting from thermolysin digestion after 0, 10, 20, and 30 min of incubation. Digestions were performed with 1 ng of thermolysin, corresponding to bar 6 in panel A. After 10 min of thermolysin digestion, lower-migrating forms of p53 were detected with PAb240, directed against an epitope in the p53 core domain. Those forms could not be detected with monoclonal antibodies directed against the p53 N terminus (PAb242) or the p53 C terminus (PAb 421). Numbers on the left in panel C are molecular masses in kilodaltons.
Figure Legend Snippet: Protease treatment of p53 activates its exonucleolytic activity. (A and B) Wild-type p53 (A) or exonuclease III (Ex. III) (B) (150 ng) was digested with various amounts of thermolysin (40 U/mg), ranging from 4 ng to 20 pg. Exonuclease activity was measured after 15 min of digestion. The amount of exonuclease III was chosen to match the exonuclease activity of 150 ng of wt p53. Incubation of p53 with buffer alone for 15 min (p53 buffer control) did not affect the p53 exonuclease activity. (C) Western blot analysis of p53 fragments resulting from thermolysin digestion after 0, 10, 20, and 30 min of incubation. Digestions were performed with 1 ng of thermolysin, corresponding to bar 6 in panel A. After 10 min of thermolysin digestion, lower-migrating forms of p53 were detected with PAb240, directed against an epitope in the p53 core domain. Those forms could not be detected with monoclonal antibodies directed against the p53 N terminus (PAb242) or the p53 C terminus (PAb 421). Numbers on the left in panel C are molecular masses in kilodaltons.

Techniques Used: Activity Assay, Incubation, Western Blot

4) Product Images from "A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA"

Article Title: A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Journal: Nucleic Acids Research

doi:

The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
Figure Legend Snippet: The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

Techniques Used: Activity Assay, Incubation, Labeling

Related Articles

High Performance Liquid Chromatography:

Article Title: Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates
Article Snippet: .. The following reagents were purchased from the sources noted: α-32 P-labeled nucleoside triphosphates (NEN or Amersham); HPLC grade nucleoside triphosphates and deoxynucleoside triphosphates (Pharmacia); Hybond N+ nylon membrane (Amersham); dinucleotide ApU, BSA, yeast Torula RNA, polymin P, and rifampicin (Sigma); M-280 streptavidin Dynabeads (Dynal, Oslo); Seq-Light (Tropix, Bedford MA); exonuclease III (Exo III) (Boehringer Mannheim); AmpliTaq (Perkin–Elmer); Ni2+ -NTA agarose (Qiagen, Chatsworth CA); phosphocellulose (Whatman); X-Omat scientific imaging film (Kodak); and 5′ biotin phosphoramidite (Glen Research, Sterling, VA). ..

Activity Assay:

Article Title: Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease
Article Snippet: .. Modulation of UDGase activity by exonuclease III (from E.coli ; Boehringer Mannheim) or LmAP was measured in the same mixture after adding from 40 to 175 U exonuclease III or a molar excess of up to 100-fold AP endonuclease. .. Pure preparations of His-tagged AP endonuclease from L.major were obtained as previously described ( ).

Article Title: Different Regulation of the p53 Core Domain Activities 3?-to-5? Exonuclease and Sequence-Specific DNA Binding
Article Snippet: .. Exonuclease III (Boehringer, Mannheim, Germany) with the same exonucleolytic activity as 150 ng of p53 was used as a control. .. Synthetic p21 promoter oligonucleotides ( ) were end labeled by using T4 polynucleotide kinase and [γ-32 P]ATP, gel purified, and used as probes in binding reactions after annealing with unlabeled complementary strand. p53 was purified by metal affinity chromatography or nuclear extraction ( ).

Imaging:

Article Title: Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates
Article Snippet: .. The following reagents were purchased from the sources noted: α-32 P-labeled nucleoside triphosphates (NEN or Amersham); HPLC grade nucleoside triphosphates and deoxynucleoside triphosphates (Pharmacia); Hybond N+ nylon membrane (Amersham); dinucleotide ApU, BSA, yeast Torula RNA, polymin P, and rifampicin (Sigma); M-280 streptavidin Dynabeads (Dynal, Oslo); Seq-Light (Tropix, Bedford MA); exonuclease III (Exo III) (Boehringer Mannheim); AmpliTaq (Perkin–Elmer); Ni2+ -NTA agarose (Qiagen, Chatsworth CA); phosphocellulose (Whatman); X-Omat scientific imaging film (Kodak); and 5′ biotin phosphoramidite (Glen Research, Sterling, VA). ..

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    Boehringer Mannheim exo iii
    <t>Exo</t> <t>III</t> assay of histone–NF-YB–NF-YC combinations. Stoichiometric amounts of the indicated combinations of HFM proteins were reconstituted with fragment 2 (labeled on the top strand [A] and bottom strand [B]), cut with Exo III for the indicated length of time, and analyzed on sequencing gels. Nuc., nucleosome.
    Exo Iii, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exo iii/product/Boehringer Mannheim
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    92
    Boehringer Mannheim exonuclease iii
    Enhancement of <t>UDGase</t> activity by exonuclease <t>III.</t> Decrease in fluorescence observed in the presence of different concentrations of exonuclease III. Open squares, UDGase; closed squares, UDGase + 40 U; triangles, UDGase +80 U; open circles, UDGase +175 U; closed circles, LmAP.
    Exonuclease Iii, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease iii/product/Boehringer Mannheim
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    Image Search Results


    Exo III assay of histone–NF-YB–NF-YC combinations. Stoichiometric amounts of the indicated combinations of HFM proteins were reconstituted with fragment 2 (labeled on the top strand [A] and bottom strand [B]), cut with Exo III for the indicated length of time, and analyzed on sequencing gels. Nuc., nucleosome.

    Journal: Molecular and Cellular Biology

    Article Title: NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms

    doi:

    Figure Lengend Snippet: Exo III assay of histone–NF-YB–NF-YC combinations. Stoichiometric amounts of the indicated combinations of HFM proteins were reconstituted with fragment 2 (labeled on the top strand [A] and bottom strand [B]), cut with Exo III for the indicated length of time, and analyzed on sequencing gels. Nuc., nucleosome.

    Article Snippet: We also performed Exo III experiments on our reconstitutions (Fig. ); this assay is based on the 3′-5′ nuclease activity of this processive enzyme, which is a measure of the stability of the protein-DNA complexes.

    Techniques: Labeling, Sequencing

    Recapitulation of the data obtained with DNase I, MNase, and Exo III assays. The X, Y, and Inr elements are boxed, and the major +1 start site of the Ea promoter is outlined. (A) DNase I. Arrows indicate the cuts in the nucleosomal region. (B) MNase. Thick arrows indicate the cuts with nucleosomes and dotted arrows with H3-H4 tetramers, thin arrows represent minor cutting sites, and brackets delimit the boundaries of the nucleosome. (C) Exo III. Thick and dotted lines indicate major stops with nucleosomes and H3-H4 tetramers, respectively. The small arrow indicates a major stop which is bypassed when NF-YB–NF-YC is reconstituted with nucleosomes. Asterisks refer to differences in the pattern observed when the NF-YB and NF-YC subunits are added to histones.

    Journal: Molecular and Cellular Biology

    Article Title: NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms

    doi:

    Figure Lengend Snippet: Recapitulation of the data obtained with DNase I, MNase, and Exo III assays. The X, Y, and Inr elements are boxed, and the major +1 start site of the Ea promoter is outlined. (A) DNase I. Arrows indicate the cuts in the nucleosomal region. (B) MNase. Thick arrows indicate the cuts with nucleosomes and dotted arrows with H3-H4 tetramers, thin arrows represent minor cutting sites, and brackets delimit the boundaries of the nucleosome. (C) Exo III. Thick and dotted lines indicate major stops with nucleosomes and H3-H4 tetramers, respectively. The small arrow indicates a major stop which is bypassed when NF-YB–NF-YC is reconstituted with nucleosomes. Asterisks refer to differences in the pattern observed when the NF-YB and NF-YC subunits are added to histones.

    Article Snippet: We also performed Exo III experiments on our reconstitutions (Fig. ); this assay is based on the 3′-5′ nuclease activity of this processive enzyme, which is a measure of the stability of the protein-DNA complexes.

    Techniques:

    Activities of WRNp on an unmodified, 5′-overhang DNA substrate. ( A ) Assay of WRNp helicase activity on the 5′-overhang DNA substrate. The unmodified substrate was incubated with or without WRNp (150 fmol) for 1 h at 37°C and the DNA products were analyzed on a non-denaturing 12% polyacrylamide gel. Under these non-denaturing electrophoretic conditions, migration of double-stranded DNA species is dictated by the length of the unlabeled strand (data not shown) and thus WRNp exonuclease degradation products cannot be resolved by this method. As a marker for the migration of the labeled strand, the DNA substrate was loaded onto the gel immediately following heat denaturation (denoted by the triangle). The positions of the intact double-stranded substrate and the labeled single-stranded 32mer are denoted to the left. ( B ) Assay of exo III, Klenow and WRNp 3′→5′ exonuclease activities on the unmodified, 5′-overhang DNA substrate. The unmodified DNA substrate (depicted at the top) was incubated without enzyme (–enzyme) or with exo III (1 U), Klenow (2 U) or WRNp (120 fmol) at 37°C for 1 h. After heating at 90°C, the single-stranded DNA products were separated on a 15% denaturing polyacrylamide gel and visualized by phosphorimaging. The identities of the 3′-end nucleotide for each single-stranded degradation product are denoted to the right.

    Journal: Nucleic Acids Research

    Article Title: Selective blockage of the 3?- > 5? exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA

    doi:

    Figure Lengend Snippet: Activities of WRNp on an unmodified, 5′-overhang DNA substrate. ( A ) Assay of WRNp helicase activity on the 5′-overhang DNA substrate. The unmodified substrate was incubated with or without WRNp (150 fmol) for 1 h at 37°C and the DNA products were analyzed on a non-denaturing 12% polyacrylamide gel. Under these non-denaturing electrophoretic conditions, migration of double-stranded DNA species is dictated by the length of the unlabeled strand (data not shown) and thus WRNp exonuclease degradation products cannot be resolved by this method. As a marker for the migration of the labeled strand, the DNA substrate was loaded onto the gel immediately following heat denaturation (denoted by the triangle). The positions of the intact double-stranded substrate and the labeled single-stranded 32mer are denoted to the left. ( B ) Assay of exo III, Klenow and WRNp 3′→5′ exonuclease activities on the unmodified, 5′-overhang DNA substrate. The unmodified DNA substrate (depicted at the top) was incubated without enzyme (–enzyme) or with exo III (1 U), Klenow (2 U) or WRNp (120 fmol) at 37°C for 1 h. After heating at 90°C, the single-stranded DNA products were separated on a 15% denaturing polyacrylamide gel and visualized by phosphorimaging. The identities of the 3′-end nucleotide for each single-stranded degradation product are denoted to the right.

    Article Snippet: Exo III and Klenow were purchased from Boehringer Mannheim (Indianapolis, IN) and T4 polynucleotide kinase was from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation, Migration, Marker, Labeling

    The 3′→5′ exonuclease activities of WRNp, exo III and Klenow on substrates containing 8-oxoG, 8-oxoA, an apurinic site or a cholesterol adduct. The DNA substrates were as depicted in Figure 2B, except that 8-oxoG, 8-oxoA, an apurinic site or cholesterol was substituted for a normal nucleotide at a defined position. The sequence and base pairing structure near the 5′-overhang end of each substrate is shown at the top. The hydroxyl groups at position 8 of guanine and adenine are denoted by ° above the modified nucleotide and the cholesterol moiety is indicated by X. For each substrate, the abnormal base pair is capitalized and in bold and the modified nucleotide is underlined, with the exception of the apurinic site, which is indicated by ^. Exonuclease reactions and subsequent analysis were carried out as in Figure 2B. For each substrate, the position of the degradation product having the modified nucleotide at the 3′-terminus of the labeled strand is denoted with arrows.

    Journal: Nucleic Acids Research

    Article Title: Selective blockage of the 3?- > 5? exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA

    doi:

    Figure Lengend Snippet: The 3′→5′ exonuclease activities of WRNp, exo III and Klenow on substrates containing 8-oxoG, 8-oxoA, an apurinic site or a cholesterol adduct. The DNA substrates were as depicted in Figure 2B, except that 8-oxoG, 8-oxoA, an apurinic site or cholesterol was substituted for a normal nucleotide at a defined position. The sequence and base pairing structure near the 5′-overhang end of each substrate is shown at the top. The hydroxyl groups at position 8 of guanine and adenine are denoted by ° above the modified nucleotide and the cholesterol moiety is indicated by X. For each substrate, the abnormal base pair is capitalized and in bold and the modified nucleotide is underlined, with the exception of the apurinic site, which is indicated by ^. Exonuclease reactions and subsequent analysis were carried out as in Figure 2B. For each substrate, the position of the degradation product having the modified nucleotide at the 3′-terminus of the labeled strand is denoted with arrows.

    Article Snippet: Exo III and Klenow were purchased from Boehringer Mannheim (Indianapolis, IN) and T4 polynucleotide kinase was from New England Biolabs (Beverly, MA).

    Techniques: Sequencing, Modification, Labeling

    The 3′→5′ exonuclease activities of WRNp, exo III and Klenow on substrates containing uracil, hypoxanthine or ethenoadenine. The DNA substrates were as depicted in Figure 2B, except that uracil, hypoxanthine or ethenoadenine (ethenoA) was substituted for a normal nucleotide at a defined position. The sequence and base pairing structure near the 5′-overhang end of each substrate is shown at the top. For each substrate, the abnormal base pair is capitalized and in bold and the modified nucleotide is underlined, with U, H and E representing uracil, hypoxanthine and ethenoadenine, respectively. Exonuclease reactions and analysis of DNA products were carried out as described in Figure 2B. For each substrate, the position of the degradation product having the modified nucleotide at the 3′-terminus of the labeled strand is denoted with arrows.

    Journal: Nucleic Acids Research

    Article Title: Selective blockage of the 3?- > 5? exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA

    doi:

    Figure Lengend Snippet: The 3′→5′ exonuclease activities of WRNp, exo III and Klenow on substrates containing uracil, hypoxanthine or ethenoadenine. The DNA substrates were as depicted in Figure 2B, except that uracil, hypoxanthine or ethenoadenine (ethenoA) was substituted for a normal nucleotide at a defined position. The sequence and base pairing structure near the 5′-overhang end of each substrate is shown at the top. For each substrate, the abnormal base pair is capitalized and in bold and the modified nucleotide is underlined, with U, H and E representing uracil, hypoxanthine and ethenoadenine, respectively. Exonuclease reactions and analysis of DNA products were carried out as described in Figure 2B. For each substrate, the position of the degradation product having the modified nucleotide at the 3′-terminus of the labeled strand is denoted with arrows.

    Article Snippet: Exo III and Klenow were purchased from Boehringer Mannheim (Indianapolis, IN) and T4 polynucleotide kinase was from New England Biolabs (Beverly, MA).

    Techniques: Sequencing, Modification, Labeling

    Enhancement of UDGase activity by exonuclease III. Decrease in fluorescence observed in the presence of different concentrations of exonuclease III. Open squares, UDGase; closed squares, UDGase + 40 U; triangles, UDGase +80 U; open circles, UDGase +175 U; closed circles, LmAP.

    Journal: Nucleic Acids Research

    Article Title: Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

    doi:

    Figure Lengend Snippet: Enhancement of UDGase activity by exonuclease III. Decrease in fluorescence observed in the presence of different concentrations of exonuclease III. Open squares, UDGase; closed squares, UDGase + 40 U; triangles, UDGase +80 U; open circles, UDGase +175 U; closed circles, LmAP.

    Article Snippet: Modulation of UDGase activity by exonuclease III (from E.coli ; Boehringer Mannheim) or LmAP was measured in the same mixture after adding from 40 to 175 U exonuclease III or a molar excess of up to 100-fold AP endonuclease.

    Techniques: Activity Assay, Fluorescence

    Diagram of experimental design and intrinsic terminator RNA secondary structures. ( A ) Transcription was initiated at the promoter (bent rightward arrow) and the ternary complexes were walked proximal to the termination site (T) on double-stranded PCR-generated templates that were biotinylated (shaded circle containing the letter B) at one of the 5′ ends. Next, the stalled elongation complexes (shaded ellipse) were digested with Exo III (shaded circles, missing a piece) and then all four NTPs were added, allowing the stalled elongation complex to resume transcript elongation. 5′ and 3′ ends of the DNA are as indicated. ( B ) The RNA secondary structures of the three intrinsic terminators, λ tR2, T 7 Te, and rrn BT1, are shown. The underlined bases indicate the positions of transcript release and the length of the RNA product [λ tR2 and rrn , T. Kerppola and M.J.C., unpublished results) and T 7 )]. The RNA secondary structures are drawn to conform to the classical model of transcript termination, with a G+C-rich stem and U-rich region immediately downstream. It should be noted, however, that for the λ tR2 and the rrn BT1 terminators, the hairpin can be extended to include upstream adenosines and downstream uridines.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates

    doi:

    Figure Lengend Snippet: Diagram of experimental design and intrinsic terminator RNA secondary structures. ( A ) Transcription was initiated at the promoter (bent rightward arrow) and the ternary complexes were walked proximal to the termination site (T) on double-stranded PCR-generated templates that were biotinylated (shaded circle containing the letter B) at one of the 5′ ends. Next, the stalled elongation complexes (shaded ellipse) were digested with Exo III (shaded circles, missing a piece) and then all four NTPs were added, allowing the stalled elongation complex to resume transcript elongation. 5′ and 3′ ends of the DNA are as indicated. ( B ) The RNA secondary structures of the three intrinsic terminators, λ tR2, T 7 Te, and rrn BT1, are shown. The underlined bases indicate the positions of transcript release and the length of the RNA product [λ tR2 and rrn , T. Kerppola and M.J.C., unpublished results) and T 7 )]. The RNA secondary structures are drawn to conform to the classical model of transcript termination, with a G+C-rich stem and U-rich region immediately downstream. It should be noted, however, that for the λ tR2 and the rrn BT1 terminators, the hairpin can be extended to include upstream adenosines and downstream uridines.

    Article Snippet: The following reagents were purchased from the sources noted: α-32 P-labeled nucleoside triphosphates (NEN or Amersham); HPLC grade nucleoside triphosphates and deoxynucleoside triphosphates (Pharmacia); Hybond N+ nylon membrane (Amersham); dinucleotide ApU, BSA, yeast Torula RNA, polymin P, and rifampicin (Sigma); M-280 streptavidin Dynabeads (Dynal, Oslo); Seq-Light (Tropix, Bedford MA); exonuclease III (Exo III) (Boehringer Mannheim); AmpliTaq (Perkin–Elmer); Ni2+ -NTA agarose (Qiagen, Chatsworth CA); phosphocellulose (Whatman); X-Omat scientific imaging film (Kodak); and 5′ biotin phosphoramidite (Glen Research, Sterling, VA).

    Techniques: Polymerase Chain Reaction, Generated

    Assaying for intrinsic termination at the λ tR2 terminator on both ss- and dsDNA templates. ( A ) Stalled C 47 complexes (lanes 1 and 5) in TGK-B 40 M 1 were chased with 500 μM ATP, 500 μM GTP, 500 μM CTP, and 500 μM UTP for 10 min at 42°C in the presence of rifampicin at 20 μg/ml (lanes 2–4 and 6–8). Lanes 2 and 6 are aliquots of the chase reactions before the pellet was separated from the supernatant fluid, lanes 3 and 7 are the RNAs associated with the Dynabeads (P), and lanes 4 and 8 are the RNAs released into the supernatant fluid (S). The λ tR2 terminator is at positions +104 to +105 relative to the transcription start site, whereas the end of the DNA template or run-off occurs at position +177. Lanes: 1–4, no Exo III; 5–8, digested with Exo III (2,000 units/ml) for 5 min at 30°C. Quantitation of the total counts within a lane between the C 47 ). ( B ) Exo III-digested DNA was analyzed on a 6% denaturing PAGE gel and detected by chemiluminescence. The nontemplate strand was end-labeled with on the 5′ end with a biotin moiety. Lanes: 1, no Exo III; 2, Exo III at 2,000 units/ml for 5 min at 30°C. The initial DNA template, 315 nucleotides long was reduced to approximately 204 nucleotides after Exo III digestion.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates

    doi:

    Figure Lengend Snippet: Assaying for intrinsic termination at the λ tR2 terminator on both ss- and dsDNA templates. ( A ) Stalled C 47 complexes (lanes 1 and 5) in TGK-B 40 M 1 were chased with 500 μM ATP, 500 μM GTP, 500 μM CTP, and 500 μM UTP for 10 min at 42°C in the presence of rifampicin at 20 μg/ml (lanes 2–4 and 6–8). Lanes 2 and 6 are aliquots of the chase reactions before the pellet was separated from the supernatant fluid, lanes 3 and 7 are the RNAs associated with the Dynabeads (P), and lanes 4 and 8 are the RNAs released into the supernatant fluid (S). The λ tR2 terminator is at positions +104 to +105 relative to the transcription start site, whereas the end of the DNA template or run-off occurs at position +177. Lanes: 1–4, no Exo III; 5–8, digested with Exo III (2,000 units/ml) for 5 min at 30°C. Quantitation of the total counts within a lane between the C 47 ). ( B ) Exo III-digested DNA was analyzed on a 6% denaturing PAGE gel and detected by chemiluminescence. The nontemplate strand was end-labeled with on the 5′ end with a biotin moiety. Lanes: 1, no Exo III; 2, Exo III at 2,000 units/ml for 5 min at 30°C. The initial DNA template, 315 nucleotides long was reduced to approximately 204 nucleotides after Exo III digestion.

    Article Snippet: The following reagents were purchased from the sources noted: α-32 P-labeled nucleoside triphosphates (NEN or Amersham); HPLC grade nucleoside triphosphates and deoxynucleoside triphosphates (Pharmacia); Hybond N+ nylon membrane (Amersham); dinucleotide ApU, BSA, yeast Torula RNA, polymin P, and rifampicin (Sigma); M-280 streptavidin Dynabeads (Dynal, Oslo); Seq-Light (Tropix, Bedford MA); exonuclease III (Exo III) (Boehringer Mannheim); AmpliTaq (Perkin–Elmer); Ni2+ -NTA agarose (Qiagen, Chatsworth CA); phosphocellulose (Whatman); X-Omat scientific imaging film (Kodak); and 5′ biotin phosphoramidite (Glen Research, Sterling, VA).

    Techniques: Quantitation Assay, Polyacrylamide Gel Electrophoresis, Labeling