escherichia coli exonuclease New England Biolabs Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs new england biolabs exonuclease i
    <t>DNA</t> with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli <t>ExoI.</t> The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    New England Biolabs Exonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 12866 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/new england biolabs exonuclease i/product/New England Biolabs
    Average 99 stars, based on 12866 article reviews
    Price from $9.99 to $1999.99
    new england biolabs exonuclease i - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs escherichia coli dna polymerase i
    Primer extension activity of exonuclease-deficient Klenow fragment of <t>DNA</t> <t>polymerase</t> I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.
    Escherichia Coli Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli dna polymerase i/product/New England Biolabs
    Average 99 stars, based on 262 article reviews
    Price from $9.99 to $1999.99
    escherichia coli dna polymerase i - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs e coli exonuclease iii
    <t>DNA</t> repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and <t>(iii)</t> the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.
    E Coli Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli exonuclease iii/product/New England Biolabs
    Average 99 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    e coli exonuclease iii - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs bal31 nuclease
    <t>DNA</t> repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and <t>(iii)</t> the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.
    Bal31 Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bal31 nuclease/product/New England Biolabs
    Average 99 stars, based on 107 article reviews
    Price from $9.99 to $1999.99
    bal31 nuclease - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs t5 exonuclease
    <t>DNA</t> repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and <t>(iii)</t> the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t5 exonuclease/product/New England Biolabs
    Average 99 stars, based on 523 article reviews
    Price from $9.99 to $1999.99
    t5 exonuclease - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs 10x exonuclease i reaction buffer
    <t>DNA</t> repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and <t>(iii)</t> the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.
    10x Exonuclease I Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/10x exonuclease i reaction buffer/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    10x exonuclease i reaction buffer - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs λ exonuclease
    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with <t>λ</t> exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).
    λ Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/λ exonuclease/product/New England Biolabs
    Average 99 stars, based on 250 article reviews
    Price from $9.99 to $1999.99
    λ exonuclease - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs t7 exonuclease
    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with <t>T7</t> exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.
    T7 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 exonuclease/product/New England Biolabs
    Average 99 stars, based on 182 article reviews
    Price from $9.99 to $1999.99
    t7 exonuclease - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Journal: Nature protocols

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq

    doi: 10.1038/s41596-018-0055-0

    Figure Lengend Snippet: Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Article Snippet: M0293L) Lambda Exonuclease (New England BioLabs, cat.no.

    Techniques:

    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Journal: PLoS ONE

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    doi: 10.1371/journal.pone.0016925

    Figure Lengend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Article Snippet: In cases where the COLIGO was still contaminated by > 5% of the linear oligonucleotide after elution (as determined by gel staining), an Exonuclease I (NEB) digest was done.

    Techniques: Polyacrylamide Gel Electrophoresis

    Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.

    Journal: Nucleic Acids Research

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair

    doi: 10.1093/nar/gkw411

    Figure Lengend Snippet: Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.

    Article Snippet: ExoI and Ssb were purchased from New England Biolabs (Ipswich, USA) and Promega (Madison, USA), respectively.

    Techniques: Agarose Gel Electrophoresis

    Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.

    Journal: Nucleic Acids Research

    Article Title: DNA-protein cross-linking by trans-[PtCl2(E-iminoether)2]. A concept for activation of the trans geometry in platinum antitumor complexes

    doi: 10.1093/nar/gkg863

    Figure Lengend Snippet: Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.

    Article Snippet: T4 DNA ligase, the Klenow fragment from DNA polymerase I (exonuclease minus, mutated to remove the 3′→5′ proofreading domain) (KF– ), restriction endonuclease EcoRI and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Inhibition, DNA Synthesis

    Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ) ( A ) and RT HIV-1 ( B ) using the 8mer/40mer and 17mer/30mer primer/template duplexes, respectively. The experiments were conducted for 30 min using undamaged templates (lanes 1), undamaged templates to which histone H1 was added at a molar ratio of 4:1 (lanes 2), the templates containing monofunctional adduct of trans-EE (lanes 3) and monofunctional adduct of trans-EE cross-linked to histone H1 (lanes 4). The pause sites opposite the platinated guanines and flanking residues are marked 19, 20, 21 and 22 (the sites opposite the platinated residue are still marked ‘Pt’). The nucleotide sequences of the templates and the primers are shown beneath the gels.

    Journal: Nucleic Acids Research

    Article Title: DNA-protein cross-linking by trans-[PtCl2(E-iminoether)2]. A concept for activation of the trans geometry in platinum antitumor complexes

    doi: 10.1093/nar/gkg863

    Figure Lengend Snippet: Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ) ( A ) and RT HIV-1 ( B ) using the 8mer/40mer and 17mer/30mer primer/template duplexes, respectively. The experiments were conducted for 30 min using undamaged templates (lanes 1), undamaged templates to which histone H1 was added at a molar ratio of 4:1 (lanes 2), the templates containing monofunctional adduct of trans-EE (lanes 3) and monofunctional adduct of trans-EE cross-linked to histone H1 (lanes 4). The pause sites opposite the platinated guanines and flanking residues are marked 19, 20, 21 and 22 (the sites opposite the platinated residue are still marked ‘Pt’). The nucleotide sequences of the templates and the primers are shown beneath the gels.

    Article Snippet: T4 DNA ligase, the Klenow fragment from DNA polymerase I (exonuclease minus, mutated to remove the 3′→5′ proofreading domain) (KF– ), restriction endonuclease EcoRI and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay

    DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Journal: Nucleic Acids Research

    Article Title: Linking uracil base excision repair and 5-fluorouracil toxicity in yeast

    doi: 10.1093/nar/gkj430

    Figure Lengend Snippet: DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Article Snippet: Briefly, 4 µg of each DNA sample was digested with E.coli exonuclease III (145 U; New England Biolabs) for 1 min at 37°C, 100 mM putrescine at 37°C for 30 min (Acros Organics), both exonuclease III and putrescine, or left undigested.

    Techniques: Derivative Assay, Knock-Out

    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Journal: Nature Communications

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    doi: 10.1038/ncomms15335

    Figure Lengend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Article Snippet: Subsequently, 1 μl Exonuclease I (NEB, M0293S), 1 μl Exonuclease III (NEB, M0206S) and 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) were added into the reaction and jointly incubated at 37 °C for 1 h. Then the mixture was purified with MinElute Reaction Cleanup Kit (3 × ERC) (QIAGEN) and its final concentration was calibrated using Qubit ssDNA Assay Kit.

    Techniques: Mutagenesis, Two Tailed Test

    Proliferation-inducing capacity of pPCR102-2 and derivatives in HUVECs. HUVECs were transiently transfected with either pPCR102-2 ( A ), pND-A8 ( B ), pND-A2 ( C ) or pPI1-His ( D ) in order to assess their proliferation-inducing potential. Forty-eight hours post transfection, the cell populations were transduced with a GFP-encoding oncoretroviral vector, which exclusively targets proliferating cells. Forty-eight hours post-transduction, GFP-mediated fluorescence was quantified by FACS. Fluorescence values were calculated by multiplying the number of GFP-expressing cells by the average intensity of GFP expression. The relative fluorescence units were obtained by comparison with GFP-mediated fluorescence control populations (cntrl) transfected with isogenic pcDNA3.1/V5-His-TOPO. Corresponding FACS histograms are also shown. All values are representative of at least three independent experiments. FS, forward scatter; FL, fluoresence.

    Journal: Nucleic Acids Research

    Article Title: Identification of a novel proliferation-inducing determinant using lentiviral expression cloning

    doi: 10.1093/nar/gng115

    Figure Lengend Snippet: Proliferation-inducing capacity of pPCR102-2 and derivatives in HUVECs. HUVECs were transiently transfected with either pPCR102-2 ( A ), pND-A8 ( B ), pND-A2 ( C ) or pPI1-His ( D ) in order to assess their proliferation-inducing potential. Forty-eight hours post transfection, the cell populations were transduced with a GFP-encoding oncoretroviral vector, which exclusively targets proliferating cells. Forty-eight hours post-transduction, GFP-mediated fluorescence was quantified by FACS. Fluorescence values were calculated by multiplying the number of GFP-expressing cells by the average intensity of GFP expression. The relative fluorescence units were obtained by comparison with GFP-mediated fluorescence control populations (cntrl) transfected with isogenic pcDNA3.1/V5-His-TOPO. Corresponding FACS histograms are also shown. All values are representative of at least three independent experiments. FS, forward scatter; FL, fluoresence.

    Article Snippet: All PCR reactions were conducted using DyNAzyme.EXT polymerase (Finnzymes, Oulu, Finland). pND-A2 and pND-A8 represent nested deletions of pPCR102-2 which were performed by restricting pPCR102-2 with KpnI and BamHI followed by Exonuclease III (NEB, Beverly, MA) mediated digestion at 37°C for 1, 2 and 3 min, respectively.

    Techniques: Transfection, Transduction, Plasmid Preparation, Fluorescence, FACS, Expressing

    Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.

    Journal: Nucleic Acids Research

    Article Title: Attenuation of DNA charge transport by compaction into a nucleosome core particle

    doi: 10.1093/nar/gkl030

    Figure Lengend Snippet: Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.

    Article Snippet: T4 Polynucleotide Kinase (PNK), T4 DNA Ligase (T4 Lig) and Exonuclease III (ExoIII) were purchased from New England Biolabs.

    Techniques: Footprinting, Activity Assay

    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).

    Journal: Analytical biochemistry

    Article Title: Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA

    doi: 10.1016/j.ab.2008.06.037

    Figure Lengend Snippet: (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).

    Article Snippet: A mixture of supercoiled (3.7 µg) and linear (3.3 µg) DNA was incubated with λ exonuclease (1 µL, 5 units/µL) and RecJf (3 µL, 30 units/µL) in a total volume of 100 µL containing 1X λ exonuclease buffer (New England Biolabs) at 37 °C for 16 h. The nicked-circular DNA preparation (14 µg) containing traces of linear DNA was then incubated again with λ exonuclease (0.5 µL) and RecJf (3 µL) in a total volume of 100 µL containing 1X λ exonuclease buffer at 37 °C for 16 h. Next, the λ exonuclease was heat-inactivated (65 °C, 10 min), and the reaction mixture was extracted with phenol and chloroform:amyl alcohol (24:1, v/v).

    Techniques: Agarose Gel Electrophoresis, Incubation, Marker

    Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.

    Journal: Analytical biochemistry

    Article Title: Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA

    doi: 10.1016/j.ab.2008.06.037

    Figure Lengend Snippet: Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.

    Article Snippet: A mixture of supercoiled (3.7 µg) and linear (3.3 µg) DNA was incubated with λ exonuclease (1 µL, 5 units/µL) and RecJf (3 µL, 30 units/µL) in a total volume of 100 µL containing 1X λ exonuclease buffer (New England Biolabs) at 37 °C for 16 h. The nicked-circular DNA preparation (14 µg) containing traces of linear DNA was then incubated again with λ exonuclease (0.5 µL) and RecJf (3 µL) in a total volume of 100 µL containing 1X λ exonuclease buffer at 37 °C for 16 h. Next, the λ exonuclease was heat-inactivated (65 °C, 10 min), and the reaction mixture was extracted with phenol and chloroform:amyl alcohol (24:1, v/v).

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Marker

    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    doi: 10.1074/jbc.M109.025825

    Figure Lengend Snippet: BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Article Snippet: G-strand overhang was also measured after incubation of genomic DNA with T7 exonuclease (1 unit/μg DNA) (New England Biolabs) in NE-Buffer 4 at 25 °C for 60 s. T7 exonuclease acts in the 5′–3′ direction to remove 5′ nucleotides from duplex DNA.

    Techniques: Hybridization, Labeling, Transfection, Plasmid Preparation, Western Blot, Over Expression