λ exonuclease Search Results


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  • 99
    New England Biolabs λ exonuclease
    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with <t>λ-exonuclease</t> (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.
    λ Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher λ exonuclease fermentas digestion
    GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with <t>λ</t> exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).
    λ Exonuclease Fermentas Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Boehringer Mannheim λ exonuclease
    GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with <t>λ</t> exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).
    λ Exonuclease, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore λ exonuclease
    GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with <t>λ</t> exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).
    λ Exonuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare λ exonuclease
    Model of DNA degradation by λ exonuclease. Initiation is achieved by two methods: plug-in at the blunt-end of linear DNA and trimer ring assembly at the single-/double-stranded junction of a partial duplex. The order of processive degradation is cleavage, product release, and translocation by electrostatic attraction with concomitant melting.
    λ Exonuclease, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Brookhaven Instruments λ exonuclease
    Model of DNA degradation by λ exonuclease. Initiation is achieved by two methods: plug-in at the blunt-end of linear DNA and trimer ring assembly at the single-/double-stranded junction of a partial duplex. The order of processive degradation is cleavage, product release, and translocation by electrostatic attraction with concomitant melting.
    λ Exonuclease, supplied by Brookhaven Instruments, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher bacteriophage λ exonuclease
    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units <t>λ</t> exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
    Bacteriophage λ Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher λ exonuclease reaction buffer
    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units <t>λ</t> exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
    λ Exonuclease Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs λ exonuclease treatment
    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units <t>λ</t> exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
    λ Exonuclease Treatment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Journal: Nucleic Acids Research

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries

    doi: 10.1093/nar/gkv544

    Figure Lengend Snippet: Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Article Snippet: Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5).

    Techniques: Modification, Polyacrylamide Gel Electrophoresis, Staining

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Journal: Nature Communications

    Article Title: Simplified ChIP-exo assays

    doi: 10.1038/s41467-018-05265-7

    Figure Lengend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Article Snippet: The λ exonuclease digestion (100 µl) containing: 20 U λ exonuclease (NEB), 1 × λ exonuclease reaction buffer (NEB), 0.1% Triton-X 100, and 5% DMSO was incubated for 30 min at 37 °C; then washed with 10 mM Tris-HCl, pH 8.0 at 4 °C.

    Techniques: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Binding Assay

    Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: In Vivo, Binding Assay, Mutagenesis

    ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Ethanol Precipitation

    GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of Octamer Transcription Factors to DNA by Glucocorticoid Receptor

    doi:

    Figure Lengend Snippet: GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).

    Article Snippet: Each sample was simultaneously digested with 100 U of restriction enzyme and 15 U of λ exonuclease (Life Technologies) for 15 min at 30°C.

    Techniques: Binding Assay, Transfection, Construct, Expressing, Polymerase Chain Reaction, Sequencing, Amplification, Western Blot

    Model of DNA degradation by λ exonuclease. Initiation is achieved by two methods: plug-in at the blunt-end of linear DNA and trimer ring assembly at the single-/double-stranded junction of a partial duplex. The order of processive degradation is cleavage, product release, and translocation by electrostatic attraction with concomitant melting.

    Journal: Nucleic Acids Research

    Article Title: Allosteric ring assembly and chemo-mechanical melting by the interaction between 5′-phosphate and λ exonuclease

    doi: 10.1093/nar/gkv1150

    Figure Lengend Snippet: Model of DNA degradation by λ exonuclease. Initiation is achieved by two methods: plug-in at the blunt-end of linear DNA and trimer ring assembly at the single-/double-stranded junction of a partial duplex. The order of processive degradation is cleavage, product release, and translocation by electrostatic attraction with concomitant melting.

    Article Snippet: The cell lysate was centrifuged for 30 min at 35 000 ×g , and re-suspended in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. λ exonuclease was purified by HisTrap FF (GE Healthcare) using a buffer (50 mM Tris–HCl (pH8.0), 300 mM NaCl and 300 mM Imidazol in a gradient method. (His)6 -tag-MBP was removed by TEV protease, which remained five glycines at the N-terminus of the protein, and purified again by HisTrap FF (GE Healthcare).

    Techniques: Translocation Assay

    Single molecule FRET assay for DNA degradation. ( A ) Top and side views of the crystal structure of λ exonuclease with DNA (PDB entry 3SM4) demonstrating that two nucleotides (violet) of the 5′-end strand are pre-melted at the ss-ds junction and inserted into the active site of one subunit. ( B ) Experimental scheme, depicting the DNA and protein tilted with an angle relative to the DNA. ( C ) FRET-time trace, demonstrating how degradation time is assigned. ( D ) FRET-histogram that was obtained as shown in (B) (top: dsDNA; middle: before the degradation without Mg 2+ ; bottom: after the degradation with Mg 2+ ). ( E ) FRET-histograms obtained before (black curve) and after (red curve) a 2-min reaction with DNA substrates with (top) and without (bottom) a 5′ terminal phosphate. ( F ) The 5′ terminal phosphate is essential for the formation of a catalytically active DNA and enzyme complex. The fitted exponential growth of 5′P-DNA (black line) and 5′OH-DNA (red line) are 20 s and 403 s, respectively. Error bars denote SEM.

    Journal: Nucleic Acids Research

    Article Title: Allosteric ring assembly and chemo-mechanical melting by the interaction between 5′-phosphate and λ exonuclease

    doi: 10.1093/nar/gkv1150

    Figure Lengend Snippet: Single molecule FRET assay for DNA degradation. ( A ) Top and side views of the crystal structure of λ exonuclease with DNA (PDB entry 3SM4) demonstrating that two nucleotides (violet) of the 5′-end strand are pre-melted at the ss-ds junction and inserted into the active site of one subunit. ( B ) Experimental scheme, depicting the DNA and protein tilted with an angle relative to the DNA. ( C ) FRET-time trace, demonstrating how degradation time is assigned. ( D ) FRET-histogram that was obtained as shown in (B) (top: dsDNA; middle: before the degradation without Mg 2+ ; bottom: after the degradation with Mg 2+ ). ( E ) FRET-histograms obtained before (black curve) and after (red curve) a 2-min reaction with DNA substrates with (top) and without (bottom) a 5′ terminal phosphate. ( F ) The 5′ terminal phosphate is essential for the formation of a catalytically active DNA and enzyme complex. The fitted exponential growth of 5′P-DNA (black line) and 5′OH-DNA (red line) are 20 s and 403 s, respectively. Error bars denote SEM.

    Article Snippet: The cell lysate was centrifuged for 30 min at 35 000 ×g , and re-suspended in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. λ exonuclease was purified by HisTrap FF (GE Healthcare) using a buffer (50 mM Tris–HCl (pH8.0), 300 mM NaCl and 300 mM Imidazol in a gradient method. (His)6 -tag-MBP was removed by TEV protease, which remained five glycines at the N-terminus of the protein, and purified again by HisTrap FF (GE Healthcare).

    Techniques:

    The lack of a phosphate at a nick in the DNA substrate prevents the formation of a stable association with λ exonuclease during degradation. ( A ) Schematic of a DNA substrate without a 5′ phosphate group at a nick in the hydrolyzed strand (nick-5′OH-DNA). ( B ) Exemplary FRET time trajectory demonstrating diffusive back and forth movement of the enzyme in the absence of a 5′ phosphate in the substrate. Approximately 90% of all trajectories exhibited diffusive movements on the nick-5′OH-DNA. The blue trace is an idealized fit that is used to extract association and dissociation rates based on the HaMMy ( 25 ) algorithm. ( C ) Transition density plots (TDP) generated from the FRET value before transition (association of the enzyme) on the x-axis and the FRET value after transition (dissociation of the enzyme) on the y-axis. To extract association and dissociation constants, HaMMy ( 25 ) analysis was performed based on 161 individual FRET time trajectories.

    Journal: Nucleic Acids Research

    Article Title: Allosteric ring assembly and chemo-mechanical melting by the interaction between 5′-phosphate and λ exonuclease

    doi: 10.1093/nar/gkv1150

    Figure Lengend Snippet: The lack of a phosphate at a nick in the DNA substrate prevents the formation of a stable association with λ exonuclease during degradation. ( A ) Schematic of a DNA substrate without a 5′ phosphate group at a nick in the hydrolyzed strand (nick-5′OH-DNA). ( B ) Exemplary FRET time trajectory demonstrating diffusive back and forth movement of the enzyme in the absence of a 5′ phosphate in the substrate. Approximately 90% of all trajectories exhibited diffusive movements on the nick-5′OH-DNA. The blue trace is an idealized fit that is used to extract association and dissociation rates based on the HaMMy ( 25 ) algorithm. ( C ) Transition density plots (TDP) generated from the FRET value before transition (association of the enzyme) on the x-axis and the FRET value after transition (dissociation of the enzyme) on the y-axis. To extract association and dissociation constants, HaMMy ( 25 ) analysis was performed based on 161 individual FRET time trajectories.

    Article Snippet: The cell lysate was centrifuged for 30 min at 35 000 ×g , and re-suspended in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. λ exonuclease was purified by HisTrap FF (GE Healthcare) using a buffer (50 mM Tris–HCl (pH8.0), 300 mM NaCl and 300 mM Imidazol in a gradient method. (His)6 -tag-MBP was removed by TEV protease, which remained five glycines at the N-terminus of the protein, and purified again by HisTrap FF (GE Healthcare).

    Techniques: Generated

    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.

    Journal: Genes & Development

    Article Title: Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing

    doi:

    Figure Lengend Snippet: Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.

    Article Snippet: Aliquots of DNA were incubated with 100 units of E. coli exonuclease III or 10 units of bacteriophage λ exonuclease (either purchased from Life Technologies, Inc. or a gift of Drs. Deb Chatterjee and Per Harbury) at 37°C for 1 hr and the completeness of their digestion was confirmed by gel electrophoresis.

    Techniques: Southern Blot, Derivative Assay, Isolation, Agarose Gel Electrophoresis, Electrophoresis, Migration, Neutralization