exonuclease iii  (TaKaRa)

 
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    Name:
    Exonuclease III
    Description:
    Exonuclease III is a 3 to 5 exonuclease that removes mononucleotides from the 3 hydroxyl termini of double stranded DNA or DNA RNA hybrids Because a set number of mononucleotides are generated during each degradation double stranded DNA degradation is progressive Exonuclease III will degrade double stranded DNA containing a blunt end a 5 overhang or a nick but it will not degrade DNA with a 3 overhang containing four or more bases This 3 to 5 exonuclease can be inactivated by heating it at 65°C for 5 min Exonuclease III is supplied in a buffer of 25 mM Tris HCl pH 8 0 50 mM KCl 0 5 mM DTT and 50 glycerol
    Catalog Number:
    2170b
    Price:
    None
    Size:
    25 000 Units
    Category:
    Exonuclease III Nucleases Modifying enzymes Cloning
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    Structured Review

    TaKaRa exonuclease iii
    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease <t>III.</t> To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the <t>DNA</t> was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.
    Exonuclease III is a 3 to 5 exonuclease that removes mononucleotides from the 3 hydroxyl termini of double stranded DNA or DNA RNA hybrids Because a set number of mononucleotides are generated during each degradation double stranded DNA degradation is progressive Exonuclease III will degrade double stranded DNA containing a blunt end a 5 overhang or a nick but it will not degrade DNA with a 3 overhang containing four or more bases This 3 to 5 exonuclease can be inactivated by heating it at 65°C for 5 min Exonuclease III is supplied in a buffer of 25 mM Tris HCl pH 8 0 50 mM KCl 0 5 mM DTT and 50 glycerol
    https://www.bioz.com/result/exonuclease iii/product/TaKaRa
    Average 93 stars, based on 8 article reviews
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    exonuclease iii - by Bioz Stars, 2020-07
    93/100 stars

    Images

    1) Product Images from "In Vitro Reconstitution of the End Replication Problem"

    Article Title: In Vitro Reconstitution of the End Replication Problem

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.17.5753-5766.2001

    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.
    Figure Legend Snippet: Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

    Techniques Used: Purification, Acrylamide Gel Assay, Labeling, Migration, Derivative Assay

    2) Product Images from "Regulation of DNA nucleases by molecular crowding"

    Article Title: Regulation of DNA nucleases by molecular crowding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm445

    Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.
    Figure Legend Snippet: Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

    Techniques Used:

    3) Product Images from "Exosomes maintain cellular homeostasis by excreting harmful DNA from cells"

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    Journal: Nature Communications

    doi: 10.1038/ncomms15287

    Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.
    Figure Legend Snippet: Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

    Techniques Used: Transfection, Western Blot

    Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
    Figure Legend Snippet: Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Techniques Used: Inhibition, Mouse Assay, Injection, Plasmid Preparation, Luciferase, shRNA, Transfection, Imaging, Western Blot, Isolation, Immunofluorescence, Staining

    Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
    Figure Legend Snippet: Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Techniques Used: Transfection, Infection, Recombinant, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Titration, Generated

    Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
    Figure Legend Snippet: Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Techniques Used: Inhibition, Transfection, Western Blot, Isolation, Cell Culture, Immunofluorescence, Staining, Infection, Plasmid Preparation, Selection

    Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
    Figure Legend Snippet: Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Techniques Used: Over Expression, Infection, Plasmid Preparation, Selection, Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Expressing

    Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P
    Figure Legend Snippet: Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

    Techniques Used: Transfection, Western Blot, Immunofluorescence, Staining

    4) Product Images from "Establishment of DNA-DNA Interactions by the Cohesin Ring"

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    Journal: Cell

    doi: 10.1016/j.cell.2017.12.021

    ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.
    Figure Legend Snippet: ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.

    Techniques Used: Incubation

    Acetyl-Acceptor Lysines and the Cohesin Loader Promote Second-DNA Capture (A) Protocol 2 experiments were carried out with the indicated order of additions, demonstrating a strong preference of reaction order during second-DNA capture. (B) Protocol 1B was used to test the ability of the indicated cohesin loading cofactors to promote second-DNA capture. Recovered DNA was analyzed by agarose gel electrophoresis and quantified. (C) WT and Psm3 K106Q (KQ) cohesin was used in a protocol 2 experiment. An aliquot was taken after the first dsDNA loading incubation to confirm comparable levels of loading by carrying out the reaction in low-salt condition (15 mM NaCl) before performing second-DNA capture on ssDNA beads followed by agarose gel electrophoresis and quantification. The means and standard deviations from three independent experiments are shown in each panel. See also Figure S4 for experiments that confirm the Mis4 requirement for second-DNA capture and the contribution of the acetyl-acceptor lysines to cohesin loading onto ssDNA.
    Figure Legend Snippet: Acetyl-Acceptor Lysines and the Cohesin Loader Promote Second-DNA Capture (A) Protocol 2 experiments were carried out with the indicated order of additions, demonstrating a strong preference of reaction order during second-DNA capture. (B) Protocol 1B was used to test the ability of the indicated cohesin loading cofactors to promote second-DNA capture. Recovered DNA was analyzed by agarose gel electrophoresis and quantified. (C) WT and Psm3 K106Q (KQ) cohesin was used in a protocol 2 experiment. An aliquot was taken after the first dsDNA loading incubation to confirm comparable levels of loading by carrying out the reaction in low-salt condition (15 mM NaCl) before performing second-DNA capture on ssDNA beads followed by agarose gel electrophoresis and quantification. The means and standard deviations from three independent experiments are shown in each panel. See also Figure S4 for experiments that confirm the Mis4 requirement for second-DNA capture and the contribution of the acetyl-acceptor lysines to cohesin loading onto ssDNA.

    Techniques Used: Agarose Gel Electrophoresis, Incubation

    Mis4-Ssl3, but not Pds5-Wapl, Promote Second-DNA Capture, Related to Figure 5 (A) Protocol 1B reactions were initiated by cohesin and Mis4-Ssl3. Then the dsDNA beads were washed and increasing concentrations of Pds5-Wapl were included for second-DNA capture. A reaction in which Mis4-Ssl3 was added back is included for comparison. The graph shows quantification of recovered free ssDNA detected by agarose gel electrophoresis. (B) Protocol 1B reactions were carried out in the presence of the indicated loading cofactors as described in Figure 5 B, but 1B3B cohesin was used. The graph shows means and the range of recovered ssDNA from two independent experiments. (C) Protocol 2 reactions were carried out in the presence of the indicated loading cofactors. The gel image shows recovery of free dsDNA on the ssDNA beads, the graph reports means and standard deviations from three independent experiments. (D) Acetyl-acceptor lysines on Psm3 contribute to ssDNA loading. DNA loading reactions were carried out with the indicated cohesin complexes using dsDNA or ssDNA as substrates. The graph shows means and standard deviations of the recovered DNA from three independent experiments.
    Figure Legend Snippet: Mis4-Ssl3, but not Pds5-Wapl, Promote Second-DNA Capture, Related to Figure 5 (A) Protocol 1B reactions were initiated by cohesin and Mis4-Ssl3. Then the dsDNA beads were washed and increasing concentrations of Pds5-Wapl were included for second-DNA capture. A reaction in which Mis4-Ssl3 was added back is included for comparison. The graph shows quantification of recovered free ssDNA detected by agarose gel electrophoresis. (B) Protocol 1B reactions were carried out in the presence of the indicated loading cofactors as described in Figure 5 B, but 1B3B cohesin was used. The graph shows means and the range of recovered ssDNA from two independent experiments. (C) Protocol 2 reactions were carried out in the presence of the indicated loading cofactors. The gel image shows recovery of free dsDNA on the ssDNA beads, the graph reports means and standard deviations from three independent experiments. (D) Acetyl-acceptor lysines on Psm3 contribute to ssDNA loading. DNA loading reactions were carried out with the indicated cohesin complexes using dsDNA or ssDNA as substrates. The graph shows means and standard deviations of the recovered DNA from three independent experiments.

    Techniques Used: Agarose Gel Electrophoresis

    RPA Impacts on Sister Chromatid Cohesion Establishment In Vivo (A) Effect of the rfa1 G77E mutation or RPA overexpression (RPA OE) on sister chromatid cohesion in ctf18Δ cells. Cells were synchronized and arrested in mitosis by nocodazole treatment. Sister chromatid cohesion at the GFP-marked URA3 locus was analyzed. Western blotting confirmed RPA overexpression. At least 100 cells were scored under each condition. The graph shows means and standard deviations from three independent experiments. (B) Smc3 acetylation was analyzed in synchronized cultures from the strains above by western blotting. The acetyl-Smc3 signal, normalized to tubulin and then to the WT signal at 90 min, was quantified in three independent repeats of the experiment. The means and standard deviations are shown. (C) The cohesin loader promotes sister chromatid cohesion establishment. Sister chromatid cohesion was monitored at indicated time points following release from G1 or HU under the indicated conditions and genotypes. (D) A model for the establishment of sister chromatid cohesion at the DNA replication fork. See the Discussion for details. See also Figure S6 for supporting genetic and cell-cycle analyses that explore the role of RPA in sister chromatid cohesion establishment.
    Figure Legend Snippet: RPA Impacts on Sister Chromatid Cohesion Establishment In Vivo (A) Effect of the rfa1 G77E mutation or RPA overexpression (RPA OE) on sister chromatid cohesion in ctf18Δ cells. Cells were synchronized and arrested in mitosis by nocodazole treatment. Sister chromatid cohesion at the GFP-marked URA3 locus was analyzed. Western blotting confirmed RPA overexpression. At least 100 cells were scored under each condition. The graph shows means and standard deviations from three independent experiments. (B) Smc3 acetylation was analyzed in synchronized cultures from the strains above by western blotting. The acetyl-Smc3 signal, normalized to tubulin and then to the WT signal at 90 min, was quantified in three independent repeats of the experiment. The means and standard deviations are shown. (C) The cohesin loader promotes sister chromatid cohesion establishment. Sister chromatid cohesion was monitored at indicated time points following release from G1 or HU under the indicated conditions and genotypes. (D) A model for the establishment of sister chromatid cohesion at the DNA replication fork. See the Discussion for details. See also Figure S6 for supporting genetic and cell-cycle analyses that explore the role of RPA in sister chromatid cohesion establishment.

    Techniques Used: Recombinase Polymerase Amplification, In Vivo, Mutagenesis, Over Expression, Western Blot

    Topological but Labile ssDNA Embrace by the Cohesin Ring (A) Gel images and quantification of cohesin-loading assays using ssDNA or dsDNA as the substrate. Mis4-Ssl3 (MS) or Pds5-Wapl (PW) were added in the presence or absence of ATP. The graph shows the means and standard deviation from three independent experiments. (B) Following loading, the recovered material was challenged with NaCl and EDTA. The gel image and graph show DNA recovery at the indicated stages of the experiment. Means and standard deviations from three independent experiments are given. (C) Schematic and outcome of the dsDNA-to-ssDNA conversion experiment using E. coli exonuclease III (exoIII). Supernatant (S) and beads-bound (B) fractions were analyzed after the NaCl and EDTA chase. The graph indicates means and standard deviations from three independent experiments. (D) Specificity of second-ssDNA capture. Gel images and quantification of the protocol 1B second-DNA capture experiments in the presence of indicated ratio of nicked circular dsDNA competitor. The graph shows the means and standard deviation from three independent experiments. See also Figure S3 , showing ssDNA stimulation of the cohesin ATPase and a control that released ssDNA remains circular.
    Figure Legend Snippet: Topological but Labile ssDNA Embrace by the Cohesin Ring (A) Gel images and quantification of cohesin-loading assays using ssDNA or dsDNA as the substrate. Mis4-Ssl3 (MS) or Pds5-Wapl (PW) were added in the presence or absence of ATP. The graph shows the means and standard deviation from three independent experiments. (B) Following loading, the recovered material was challenged with NaCl and EDTA. The gel image and graph show DNA recovery at the indicated stages of the experiment. Means and standard deviations from three independent experiments are given. (C) Schematic and outcome of the dsDNA-to-ssDNA conversion experiment using E. coli exonuclease III (exoIII). Supernatant (S) and beads-bound (B) fractions were analyzed after the NaCl and EDTA chase. The graph indicates means and standard deviations from three independent experiments. (D) Specificity of second-ssDNA capture. Gel images and quantification of the protocol 1B second-DNA capture experiments in the presence of indicated ratio of nicked circular dsDNA competitor. The graph shows the means and standard deviation from three independent experiments. See also Figure S3 , showing ssDNA stimulation of the cohesin ATPase and a control that released ssDNA remains circular.

    Techniques Used: Mass Spectrometry, Standard Deviation

    Second-DNA Capture is Topological in Nature, Related to Figure 2 (A) Cohesin must topologically embrace dsDNA to mediate second-DNA capture. Protocol 1 reactions were carried out with ‘closed’ (C) or ‘linear’ (L) topology dsDNA beads. The gel image and graph show recovery of free ssDNA. The graph shows means and standard deviation from three independent experiments (WT cohesin) or the range of recovered ssDNA from two independent experiments (1B3B cohesin). (B) A DNA release experiment as shown in Figure 2 A was carried out using 1B3B cohesin. (C) Schematic of a DNA release experiment following protocol 2 s DNA capture. The ssDNA substrate was converted to dsDNA by DNA synthesis following capture. Then either of the two circular DNAs was digested with unique restriction enzymes, PstI (DNA beads) or BglII (free dsDNA). Recovered DNAs at the indicated stages of the experiment were analyzed by agarose gel electrophoresis. Input, bead bound (B) and supernatant (S) fractions are shown. (D) TEV cleavage of cohesin following second-DNA capture using protocol 2. The gel shows a representative image of input and recovered DNA, using WT and TEV cleavable (21TEV) cohesin, without or with TEV protease (TEV) treatment. After TEV protease treatment, the beads were washed with high salt buffer and recovered DNA was analyzed. The graph depicts the means and standard deviations from three independent experiments.
    Figure Legend Snippet: Second-DNA Capture is Topological in Nature, Related to Figure 2 (A) Cohesin must topologically embrace dsDNA to mediate second-DNA capture. Protocol 1 reactions were carried out with ‘closed’ (C) or ‘linear’ (L) topology dsDNA beads. The gel image and graph show recovery of free ssDNA. The graph shows means and standard deviation from three independent experiments (WT cohesin) or the range of recovered ssDNA from two independent experiments (1B3B cohesin). (B) A DNA release experiment as shown in Figure 2 A was carried out using 1B3B cohesin. (C) Schematic of a DNA release experiment following protocol 2 s DNA capture. The ssDNA substrate was converted to dsDNA by DNA synthesis following capture. Then either of the two circular DNAs was digested with unique restriction enzymes, PstI (DNA beads) or BglII (free dsDNA). Recovered DNAs at the indicated stages of the experiment were analyzed by agarose gel electrophoresis. Input, bead bound (B) and supernatant (S) fractions are shown. (D) TEV cleavage of cohesin following second-DNA capture using protocol 2. The gel shows a representative image of input and recovered DNA, using WT and TEV cleavable (21TEV) cohesin, without or with TEV protease (TEV) treatment. After TEV protease treatment, the beads were washed with high salt buffer and recovered DNA was analyzed. The graph depicts the means and standard deviations from three independent experiments.

    Techniques Used: Standard Deviation, DNA Synthesis, Agarose Gel Electrophoresis

    ssDNA, but not dsDNA, Is a Substrate for Second-Strand Capture, Related to Figure 1 (A and B) Extended gel images of Figures 1 A and 1B, showing both beads bound and supernatant fractions. 25% of the supernatant fractions are shown. (C) Protocol 2 reactions were carried out using the indicated protein concentrations (each of cohesin, Mis4-Ssl3 and Psc3). The gel image and quantification of captured dsDNA are shown. (D) A typical gel image of inputs and products of a second-DNA capture reaction following protocol 1B, performed with WT cohesin. (E) Gel images showing second-DNA capture by 1B3B cohesin, containing Walker B mutations in both Psm1 and Psm3, using a protocol 1 reaction. (F) As (E), but the reaction followed protocol 2. (G) Competition of ATP with ADP or non-hydrolyzable ATP-γ−S. As in Figure 1 E (top gel image), but an additional reaction was performed in which 0.25 mM of ATP was present in all reactions that were then supplemented by additional nucleotides. The ability of ADP and ATP-γ−S to compete with ATP demonstrates that both nucleotides are able to bind cohesin, but that their hydrolysis is required for second-DNA capture. (H) Cohesin mediates second-DNA capture irrespective of sequence homology. Protocol 2 reactions were carried out using pSKsxAS ssDNA (partially homologous to the free dsDNA substrate) or ΦX174 virion ssDNA (of unrelated sequence) as substrates. The graph presents means and standard deviations from three independent experiments.
    Figure Legend Snippet: ssDNA, but not dsDNA, Is a Substrate for Second-Strand Capture, Related to Figure 1 (A and B) Extended gel images of Figures 1 A and 1B, showing both beads bound and supernatant fractions. 25% of the supernatant fractions are shown. (C) Protocol 2 reactions were carried out using the indicated protein concentrations (each of cohesin, Mis4-Ssl3 and Psc3). The gel image and quantification of captured dsDNA are shown. (D) A typical gel image of inputs and products of a second-DNA capture reaction following protocol 1B, performed with WT cohesin. (E) Gel images showing second-DNA capture by 1B3B cohesin, containing Walker B mutations in both Psm1 and Psm3, using a protocol 1 reaction. (F) As (E), but the reaction followed protocol 2. (G) Competition of ATP with ADP or non-hydrolyzable ATP-γ−S. As in Figure 1 E (top gel image), but an additional reaction was performed in which 0.25 mM of ATP was present in all reactions that were then supplemented by additional nucleotides. The ability of ADP and ATP-γ−S to compete with ATP demonstrates that both nucleotides are able to bind cohesin, but that their hydrolysis is required for second-DNA capture. (H) Cohesin mediates second-DNA capture irrespective of sequence homology. Protocol 2 reactions were carried out using pSKsxAS ssDNA (partially homologous to the free dsDNA substrate) or ΦX174 virion ssDNA (of unrelated sequence) as substrates. The graph presents means and standard deviations from three independent experiments.

    Techniques Used: Sequencing

    Second-DNA Capture by the Fission Yeast Cohesin Ring (A) Schematic of the second-DNA capture assay (protocol 1) and a gel image showing input and recovered DNA from the assay performed with the indicated substrates. ds, dsDNA; ss, ssDNA; rc, relaxed circular; c, circular. All reactions were carried out in the presence of ATP and an ATP regenerating system. 16.7% or 25% of input free dsDNA or ssDNA are shown. (B) Schematic of the second-DNA capture assay (protocol 2) and a representative gel image. 25% of input DNA is shown. (C and D) Quantification of the assays in (A) and (B), respectively, performed with WT and 1B3B cohesin. The means and standard deviations from three independent experiments are shown. (E) Quantification of second-DNA capture, using protocol 1B, in the absence or presence of the indicated adenosine derivatives. The means and standard deviations from three independent experiments are shown. See also Figure S1 for gel images that include supernatant fractions, reactions using 1B3B cohesin, titration of components, ATP competition, and an assay using ΦX174 ssDNA.
    Figure Legend Snippet: Second-DNA Capture by the Fission Yeast Cohesin Ring (A) Schematic of the second-DNA capture assay (protocol 1) and a gel image showing input and recovered DNA from the assay performed with the indicated substrates. ds, dsDNA; ss, ssDNA; rc, relaxed circular; c, circular. All reactions were carried out in the presence of ATP and an ATP regenerating system. 16.7% or 25% of input free dsDNA or ssDNA are shown. (B) Schematic of the second-DNA capture assay (protocol 2) and a representative gel image. 25% of input DNA is shown. (C and D) Quantification of the assays in (A) and (B), respectively, performed with WT and 1B3B cohesin. The means and standard deviations from three independent experiments are shown. (E) Quantification of second-DNA capture, using protocol 1B, in the absence or presence of the indicated adenosine derivatives. The means and standard deviations from three independent experiments are shown. See also Figure S1 for gel images that include supernatant fractions, reactions using 1B3B cohesin, titration of components, ATP competition, and an assay using ΦX174 ssDNA.

    Techniques Used: Titration

    5) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.

    Techniques Used: In Vitro, Ligation, Sequencing, Marker, Concentration Assay, Electrophoresis

    6) Product Images from "A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors"

    Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047159

    Expression ratio and duration of GFP in different eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP -MiLV. A: Average ratio of GFP+ cells in HEK 293, NIH 3T3, CNE2 cells upon transfection with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV for 48 h; B: The duration of GFP expression in HEK 293, NIH 3T3, CNE2 cells; C: The GFP fluorescence intensity AU (arbitrary units) of HEK 293 cells after transfected with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV. Data are representative of at least three experiments. *p
    Figure Legend Snippet: Expression ratio and duration of GFP in different eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP -MiLV. A: Average ratio of GFP+ cells in HEK 293, NIH 3T3, CNE2 cells upon transfection with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV for 48 h; B: The duration of GFP expression in HEK 293, NIH 3T3, CNE2 cells; C: The GFP fluorescence intensity AU (arbitrary units) of HEK 293 cells after transfected with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV. Data are representative of at least three experiments. *p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Fluorescence

    Effect of DNA-induced pro-inflammatory cytokines on GFP in the blood after intravenous injection eGFP -MiLV and pEGFP-N3 plasmid. Mice received an intravenous injection of 40 µg eGFP -MiLV and pEGFP-N3 plasmid. At 2 h after injection, the levels of TNF-α, IL-6 and IL-12 in blood were measured. The results are expressed at the mean ± SD of three mice. * p
    Figure Legend Snippet: Effect of DNA-induced pro-inflammatory cytokines on GFP in the blood after intravenous injection eGFP -MiLV and pEGFP-N3 plasmid. Mice received an intravenous injection of 40 µg eGFP -MiLV and pEGFP-N3 plasmid. At 2 h after injection, the levels of TNF-α, IL-6 and IL-12 in blood were measured. The results are expressed at the mean ± SD of three mice. * p

    Techniques Used: Injection, Plasmid Preparation, Mouse Assay

    7) Product Images from "Establishment of DNA-DNA Interactions by the Cohesin Ring"

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    Journal: Cell

    doi: 10.1016/j.cell.2017.12.021

    ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.
    Figure Legend Snippet: ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.

    Techniques Used: Incubation

    8) Product Images from "Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system"

    Article Title: Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh039

    The MALDI-TOF MS spectra for genotyping of SNP in apoE gene by using exonuclease III/nuclease S1/PNA systems. ( A ) Analysis of dsDNA from apoE 4 (250 bp) at codon 112 using PNA (S112G) . Reaction conditions: [ apoE DNA] = 3 µM,
    Figure Legend Snippet: The MALDI-TOF MS spectra for genotyping of SNP in apoE gene by using exonuclease III/nuclease S1/PNA systems. ( A ) Analysis of dsDNA from apoE 4 (250 bp) at codon 112 using PNA (S112G) . Reaction conditions: [ apoE DNA] = 3 µM,

    Techniques Used: Mass Spectrometry

    9) Product Images from "Cloning and characterization of the 5?-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase ? 68 kDa subunit gene"

    Article Title: Cloning and characterization of the 5?-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase ? 68 kDa subunit gene

    Journal: Nucleic Acids Research

    doi:

    Detection of the sites activated by E2F. ( A ) Three regions of the p68 gene promoter (+9/+16, –11/–3 and –19/–12) were mutated (the mutations were transversions of each base in the three base stretch and are indicated by X). These sites are represented by hatched boxes. Each mutant plasmid was co-transfected into NIH 3T3 cells with a plasmid carrying CMV-E2F-1 or CMV. The promoter activities are indicated as relative activity with respect to the activity of pKL12 without E2F-1. The promoter activity with and without E2F-1 is represented by open and closed bars, respectively. The increase in the relative activity of luciferase with CMV-E2F-1 is presented on the right. ( B ) The wild-type sequence and mutation sequence. Open boxes indicate three homologous E2F-binding sites and arrows represent transversion mutations.
    Figure Legend Snippet: Detection of the sites activated by E2F. ( A ) Three regions of the p68 gene promoter (+9/+16, –11/–3 and –19/–12) were mutated (the mutations were transversions of each base in the three base stretch and are indicated by X). These sites are represented by hatched boxes. Each mutant plasmid was co-transfected into NIH 3T3 cells with a plasmid carrying CMV-E2F-1 or CMV. The promoter activities are indicated as relative activity with respect to the activity of pKL12 without E2F-1. The promoter activity with and without E2F-1 is represented by open and closed bars, respectively. The increase in the relative activity of luciferase with CMV-E2F-1 is presented on the right. ( B ) The wild-type sequence and mutation sequence. Open boxes indicate three homologous E2F-binding sites and arrows represent transversion mutations.

    Techniques Used: Mutagenesis, Plasmid Preparation, Transfection, Activity Assay, Luciferase, Sequencing, Binding Assay

    10) Product Images from "Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation"

    Article Title: Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation

    Journal: Cell Research

    doi: 10.1038/cr.2012.157

    Zscan4 leads to longer telomeres in the resultant iPS cells and significantly improves the developmental potential of iPS cells. (A) TRF analysis of iPS cells and control ES cells (E14) between passages 7-10 demonstrated longer telomeres in OSKMZ-induced iPS cells compared to OSKM-induced iPS cells. (B) A histogram shows the distribution of relative telomere length (in TFUs) measured by Q-FISH in iPS cells. The median telomere length (white bars) is shown as the mean ± s.d. above each panel. The passage number was indicated. (C) Three live-born (E19.5) all-iPS cell mice generated from OSKMZ-1 cells via TCA. (D) An adult all-iPS cell mouse generated from OSKMZ-1 via TCA. (E) Genotype analyses of all-iPS cell mice. Retrovirus integration (left) and SSLP (right) analyses were performed using genomic DNA isolated from tail tips of adult mice. Genomic DNA from C57BL/6, DBA and ICR mice served as controls.
    Figure Legend Snippet: Zscan4 leads to longer telomeres in the resultant iPS cells and significantly improves the developmental potential of iPS cells. (A) TRF analysis of iPS cells and control ES cells (E14) between passages 7-10 demonstrated longer telomeres in OSKMZ-induced iPS cells compared to OSKM-induced iPS cells. (B) A histogram shows the distribution of relative telomere length (in TFUs) measured by Q-FISH in iPS cells. The median telomere length (white bars) is shown as the mean ± s.d. above each panel. The passage number was indicated. (C) Three live-born (E19.5) all-iPS cell mice generated from OSKMZ-1 cells via TCA. (D) An adult all-iPS cell mouse generated from OSKMZ-1 via TCA. (E) Genotype analyses of all-iPS cell mice. Retrovirus integration (left) and SSLP (right) analyses were performed using genomic DNA isolated from tail tips of adult mice. Genomic DNA from C57BL/6, DBA and ICR mice served as controls.

    Techniques Used: Fluorescence In Situ Hybridization, Mouse Assay, Generated, Isolation

    11) Product Images from "Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes"

    Article Title: Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes

    Journal: Particle and Fibre Toxicology

    doi: 10.1186/1743-8977-8-1

    Effects of endocytosis and NADPH oxidase inhibitor on DNA damage by silica particle treatment . Effects of endocytosis inhibitor ( A and B ) or NADPH oxidase inhibitor ( C and D ) on DNA strand breaks induced by silica particle treatment in HaCaT cells. ( A and B ) HaCaT cells were pretreated with 10 mM cytochalasin D (Cyto D) for 30 min (Cyto D + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. ( C and D ) HaCaT cells were pretreated with 40 mM apocynin (Apo) for 30 min (Apo + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. As a positive control, HaCaT cells were treated with 0.2 mM H 2 O 2 for 3 h. ( A and C ) Column height shows the tail length. ( B and D ) Column height shows the tail moment. Data shown are means ± SD of at least 16 cells per sample. Results shown are representative of more than three independent experiments. * P
    Figure Legend Snippet: Effects of endocytosis and NADPH oxidase inhibitor on DNA damage by silica particle treatment . Effects of endocytosis inhibitor ( A and B ) or NADPH oxidase inhibitor ( C and D ) on DNA strand breaks induced by silica particle treatment in HaCaT cells. ( A and B ) HaCaT cells were pretreated with 10 mM cytochalasin D (Cyto D) for 30 min (Cyto D + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. ( C and D ) HaCaT cells were pretreated with 40 mM apocynin (Apo) for 30 min (Apo + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. As a positive control, HaCaT cells were treated with 0.2 mM H 2 O 2 for 3 h. ( A and C ) Column height shows the tail length. ( B and D ) Column height shows the tail moment. Data shown are means ± SD of at least 16 cells per sample. Results shown are representative of more than three independent experiments. * P

    Techniques Used: Incubation, Positive Control

    12) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.

    Techniques Used: In Vitro, Ligation, Sequencing, Marker, Concentration Assay, Electrophoresis

    13) Product Images from "Complete sequences of Schizosaccharomyces pombe subtelomeres reveal multiple patterns of genome variation"

    Article Title: Complete sequences of Schizosaccharomyces pombe subtelomeres reveal multiple patterns of genome variation

    Journal: bioRxiv

    doi: 10.1101/2020.03.09.983726

    Sequencing of telomere-proximal SH regions by the serial deletion method. A telomere-proximal SH region with various common segments (∼5 kb) was amplified by PCR from each 972SD4 strain, and inserted into a vector. The resultant plasmid was digested with restriction enzymes, KpnI and XhoI, at the multi-cloning sites (MCSs) of the vector. The linearized plasmid was serially deleted from the 5’-protruding XhoI cutting site by treatment with exonuclease III (ExoIII) and mung bean nuclease (MBN) for fixed times. After deletion of the SH region, both ends of the plasmid were blunted by klenow fragment (KF) and ligated by DNA ligase. The resultant re-cirsularized plasmids were cloned using E. coli . The partially deleted SH region was sequenced using primers that aneal to MCSs of the vector. And the sequences were assembled using overlapping sequences.
    Figure Legend Snippet: Sequencing of telomere-proximal SH regions by the serial deletion method. A telomere-proximal SH region with various common segments (∼5 kb) was amplified by PCR from each 972SD4 strain, and inserted into a vector. The resultant plasmid was digested with restriction enzymes, KpnI and XhoI, at the multi-cloning sites (MCSs) of the vector. The linearized plasmid was serially deleted from the 5’-protruding XhoI cutting site by treatment with exonuclease III (ExoIII) and mung bean nuclease (MBN) for fixed times. After deletion of the SH region, both ends of the plasmid were blunted by klenow fragment (KF) and ligated by DNA ligase. The resultant re-cirsularized plasmids were cloned using E. coli . The partially deleted SH region was sequenced using primers that aneal to MCSs of the vector. And the sequences were assembled using overlapping sequences.

    Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay

    14) Product Images from "Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends"

    Article Title: Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0660-7

    Analysis of MEDEs using a capillary sequencer. FAM-labeled DNA samples were analyzed using a capillary sequencer after mixing with LIZ500 size standard-supplemented HiDi-formamide (HiDi-LIZ500). Two peaks of 100 and 150 nucleotides from the LIZ size standard, used to calibrate the electropherogram, are indicated by filled triangles. a H4 fraction DNA mixture before reaction. b Merged view of ten results obtained by analyzing H4 fraction DNA mixture independently. c – g Data obtained after different enzymatic treatments indicated on the left. Samples were purified using a DNA clean-up column before mixing with HiDi-LIZ500. T4DP, T4 DNA polymerase; SAP shrimp alkaline phosphatase; E3T4, combined treatments with exonuclease III and T4DP; SAP-T4DP, SAP-treated sample purified and treated with T4DP; E3T4-T4DP, E3T4-treated sample purified and treated with T4DP. In panel g , each peak is labeled with a corresponding base. All data were y-axis scaled so that sums of FAM peak areas are apparently even across the panels
    Figure Legend Snippet: Analysis of MEDEs using a capillary sequencer. FAM-labeled DNA samples were analyzed using a capillary sequencer after mixing with LIZ500 size standard-supplemented HiDi-formamide (HiDi-LIZ500). Two peaks of 100 and 150 nucleotides from the LIZ size standard, used to calibrate the electropherogram, are indicated by filled triangles. a H4 fraction DNA mixture before reaction. b Merged view of ten results obtained by analyzing H4 fraction DNA mixture independently. c – g Data obtained after different enzymatic treatments indicated on the left. Samples were purified using a DNA clean-up column before mixing with HiDi-LIZ500. T4DP, T4 DNA polymerase; SAP shrimp alkaline phosphatase; E3T4, combined treatments with exonuclease III and T4DP; SAP-T4DP, SAP-treated sample purified and treated with T4DP; E3T4-T4DP, E3T4-treated sample purified and treated with T4DP. In panel g , each peak is labeled with a corresponding base. All data were y-axis scaled so that sums of FAM peak areas are apparently even across the panels

    Techniques Used: Labeling, Purification

    Terminal deoxynucleotidyl transferase (TdT) treatment of samples pretreated with different enzymes. H4 DNA mixture containing 25 fmol of FAM-labeled DNA was subjected to different enzymatic treatments, followed by TdT treatment. The panels show the samples before treatment ( a , identical to that shown in Fig. 1a ); samples treated with TdT alone ( b ); samples treated with SAP followed by TdT ( c ); samples treated serially with SAP, T4DP, and TdT ( d ); samples treated serially with a combination of exonuclease III and T4DP, T4DP alone, and TdT ( e ). Between the two enzymatic treatments, the samples were purified using the DNA clean-up column. Following the TdT treatment, the samples were purified using a DNA clean-up column before mixing with HiDi-LIZ500. All data were y-axis scaled so that sums of FAM peak areas are apparently even across the panels. Two calibrator peaks from the LIZ size standard (100 and 300 nucleotides) are indicated by filled triangles, and relevant size marker locations are also indicated
    Figure Legend Snippet: Terminal deoxynucleotidyl transferase (TdT) treatment of samples pretreated with different enzymes. H4 DNA mixture containing 25 fmol of FAM-labeled DNA was subjected to different enzymatic treatments, followed by TdT treatment. The panels show the samples before treatment ( a , identical to that shown in Fig. 1a ); samples treated with TdT alone ( b ); samples treated with SAP followed by TdT ( c ); samples treated serially with SAP, T4DP, and TdT ( d ); samples treated serially with a combination of exonuclease III and T4DP, T4DP alone, and TdT ( e ). Between the two enzymatic treatments, the samples were purified using the DNA clean-up column. Following the TdT treatment, the samples were purified using a DNA clean-up column before mixing with HiDi-LIZ500. All data were y-axis scaled so that sums of FAM peak areas are apparently even across the panels. Two calibrator peaks from the LIZ size standard (100 and 300 nucleotides) are indicated by filled triangles, and relevant size marker locations are also indicated

    Techniques Used: Labeling, Purification, Marker

    Related Articles

    DNA Extraction:

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells
    Article Snippet: .. Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions . .. After heat inactivation, the exosomal DNA was purified by Proteinase K (Wako) treatment.

    Agarose Gel Electrophoresis:

    Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
    Article Snippet: .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis. .. Construction of pLMP1-MEKK1 -MiLV The EBV genome was extracted from B95-8 cells using phenol/chloroform and purified by ethanol precipitation.

    In Vitro:

    Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
    Article Snippet: .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis. .. Construction of pLMP1-MEKK1 -MiLV The EBV genome was extracted from B95-8 cells using phenol/chloroform and purified by ethanol precipitation.

    Ligation:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

    Isolation:

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells
    Article Snippet: .. Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions . .. After heat inactivation, the exosomal DNA was purified by Proteinase K (Wako) treatment.

    Purification:

    Article Title: In Vitro Reconstitution of the End Replication Problem
    Article Snippet: .. The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs). ..

    Incubation:

    Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
    Article Snippet: .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis. .. Construction of pLMP1-MEKK1 -MiLV The EBV genome was extracted from B95-8 cells using phenol/chloroform and purified by ethanol precipitation.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

    Plasmid Preparation:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

    Hybridization:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

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    TaKaRa exonuclease iii
    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease <t>III.</t> To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the <t>DNA</t> was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.
    Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

    Journal: Molecular and Cellular Biology

    Article Title: In Vitro Reconstitution of the End Replication Problem

    doi: 10.1128/MCB.21.17.5753-5766.2001

    Figure Lengend Snippet: Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

    Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs).

    Techniques: Purification, Acrylamide Gel Assay, Labeling, Migration, Derivative Assay

    Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

    Journal: Nucleic Acids Research

    Article Title: Regulation of DNA nucleases by molecular crowding

    doi: 10.1093/nar/gkm445

    Figure Lengend Snippet: Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

    Article Snippet: Exonuclease I from Escherichia coli , S1 nuclease from Asergillus oryzae and exonuclease III from E. coli were purchased from Takara Bio (Tokyo, Japan).

    Techniques:

    Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Western Blot

    Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Inhibition, Mouse Assay, Injection, Plasmid Preparation, Luciferase, shRNA, Transfection, Imaging, Western Blot, Isolation, Immunofluorescence, Staining

    Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Infection, Recombinant, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Titration, Generated

    Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Inhibition, Transfection, Western Blot, Isolation, Cell Culture, Immunofluorescence, Staining, Infection, Plasmid Preparation, Selection

    Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Over Expression, Infection, Plasmid Preparation, Selection, Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Expressing

    Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Western Blot, Immunofluorescence, Staining

    ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Incubation

    Acetyl-Acceptor Lysines and the Cohesin Loader Promote Second-DNA Capture (A) Protocol 2 experiments were carried out with the indicated order of additions, demonstrating a strong preference of reaction order during second-DNA capture. (B) Protocol 1B was used to test the ability of the indicated cohesin loading cofactors to promote second-DNA capture. Recovered DNA was analyzed by agarose gel electrophoresis and quantified. (C) WT and Psm3 K106Q (KQ) cohesin was used in a protocol 2 experiment. An aliquot was taken after the first dsDNA loading incubation to confirm comparable levels of loading by carrying out the reaction in low-salt condition (15 mM NaCl) before performing second-DNA capture on ssDNA beads followed by agarose gel electrophoresis and quantification. The means and standard deviations from three independent experiments are shown in each panel. See also Figure S4 for experiments that confirm the Mis4 requirement for second-DNA capture and the contribution of the acetyl-acceptor lysines to cohesin loading onto ssDNA.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Acetyl-Acceptor Lysines and the Cohesin Loader Promote Second-DNA Capture (A) Protocol 2 experiments were carried out with the indicated order of additions, demonstrating a strong preference of reaction order during second-DNA capture. (B) Protocol 1B was used to test the ability of the indicated cohesin loading cofactors to promote second-DNA capture. Recovered DNA was analyzed by agarose gel electrophoresis and quantified. (C) WT and Psm3 K106Q (KQ) cohesin was used in a protocol 2 experiment. An aliquot was taken after the first dsDNA loading incubation to confirm comparable levels of loading by carrying out the reaction in low-salt condition (15 mM NaCl) before performing second-DNA capture on ssDNA beads followed by agarose gel electrophoresis and quantification. The means and standard deviations from three independent experiments are shown in each panel. See also Figure S4 for experiments that confirm the Mis4 requirement for second-DNA capture and the contribution of the acetyl-acceptor lysines to cohesin loading onto ssDNA.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Agarose Gel Electrophoresis, Incubation

    Mis4-Ssl3, but not Pds5-Wapl, Promote Second-DNA Capture, Related to Figure 5 (A) Protocol 1B reactions were initiated by cohesin and Mis4-Ssl3. Then the dsDNA beads were washed and increasing concentrations of Pds5-Wapl were included for second-DNA capture. A reaction in which Mis4-Ssl3 was added back is included for comparison. The graph shows quantification of recovered free ssDNA detected by agarose gel electrophoresis. (B) Protocol 1B reactions were carried out in the presence of the indicated loading cofactors as described in Figure 5 B, but 1B3B cohesin was used. The graph shows means and the range of recovered ssDNA from two independent experiments. (C) Protocol 2 reactions were carried out in the presence of the indicated loading cofactors. The gel image shows recovery of free dsDNA on the ssDNA beads, the graph reports means and standard deviations from three independent experiments. (D) Acetyl-acceptor lysines on Psm3 contribute to ssDNA loading. DNA loading reactions were carried out with the indicated cohesin complexes using dsDNA or ssDNA as substrates. The graph shows means and standard deviations of the recovered DNA from three independent experiments.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Mis4-Ssl3, but not Pds5-Wapl, Promote Second-DNA Capture, Related to Figure 5 (A) Protocol 1B reactions were initiated by cohesin and Mis4-Ssl3. Then the dsDNA beads were washed and increasing concentrations of Pds5-Wapl were included for second-DNA capture. A reaction in which Mis4-Ssl3 was added back is included for comparison. The graph shows quantification of recovered free ssDNA detected by agarose gel electrophoresis. (B) Protocol 1B reactions were carried out in the presence of the indicated loading cofactors as described in Figure 5 B, but 1B3B cohesin was used. The graph shows means and the range of recovered ssDNA from two independent experiments. (C) Protocol 2 reactions were carried out in the presence of the indicated loading cofactors. The gel image shows recovery of free dsDNA on the ssDNA beads, the graph reports means and standard deviations from three independent experiments. (D) Acetyl-acceptor lysines on Psm3 contribute to ssDNA loading. DNA loading reactions were carried out with the indicated cohesin complexes using dsDNA or ssDNA as substrates. The graph shows means and standard deviations of the recovered DNA from three independent experiments.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Agarose Gel Electrophoresis

    RPA Impacts on Sister Chromatid Cohesion Establishment In Vivo (A) Effect of the rfa1 G77E mutation or RPA overexpression (RPA OE) on sister chromatid cohesion in ctf18Δ cells. Cells were synchronized and arrested in mitosis by nocodazole treatment. Sister chromatid cohesion at the GFP-marked URA3 locus was analyzed. Western blotting confirmed RPA overexpression. At least 100 cells were scored under each condition. The graph shows means and standard deviations from three independent experiments. (B) Smc3 acetylation was analyzed in synchronized cultures from the strains above by western blotting. The acetyl-Smc3 signal, normalized to tubulin and then to the WT signal at 90 min, was quantified in three independent repeats of the experiment. The means and standard deviations are shown. (C) The cohesin loader promotes sister chromatid cohesion establishment. Sister chromatid cohesion was monitored at indicated time points following release from G1 or HU under the indicated conditions and genotypes. (D) A model for the establishment of sister chromatid cohesion at the DNA replication fork. See the Discussion for details. See also Figure S6 for supporting genetic and cell-cycle analyses that explore the role of RPA in sister chromatid cohesion establishment.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: RPA Impacts on Sister Chromatid Cohesion Establishment In Vivo (A) Effect of the rfa1 G77E mutation or RPA overexpression (RPA OE) on sister chromatid cohesion in ctf18Δ cells. Cells were synchronized and arrested in mitosis by nocodazole treatment. Sister chromatid cohesion at the GFP-marked URA3 locus was analyzed. Western blotting confirmed RPA overexpression. At least 100 cells were scored under each condition. The graph shows means and standard deviations from three independent experiments. (B) Smc3 acetylation was analyzed in synchronized cultures from the strains above by western blotting. The acetyl-Smc3 signal, normalized to tubulin and then to the WT signal at 90 min, was quantified in three independent repeats of the experiment. The means and standard deviations are shown. (C) The cohesin loader promotes sister chromatid cohesion establishment. Sister chromatid cohesion was monitored at indicated time points following release from G1 or HU under the indicated conditions and genotypes. (D) A model for the establishment of sister chromatid cohesion at the DNA replication fork. See the Discussion for details. See also Figure S6 for supporting genetic and cell-cycle analyses that explore the role of RPA in sister chromatid cohesion establishment.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Recombinase Polymerase Amplification, In Vivo, Mutagenesis, Over Expression, Western Blot

    Topological but Labile ssDNA Embrace by the Cohesin Ring (A) Gel images and quantification of cohesin-loading assays using ssDNA or dsDNA as the substrate. Mis4-Ssl3 (MS) or Pds5-Wapl (PW) were added in the presence or absence of ATP. The graph shows the means and standard deviation from three independent experiments. (B) Following loading, the recovered material was challenged with NaCl and EDTA. The gel image and graph show DNA recovery at the indicated stages of the experiment. Means and standard deviations from three independent experiments are given. (C) Schematic and outcome of the dsDNA-to-ssDNA conversion experiment using E. coli exonuclease III (exoIII). Supernatant (S) and beads-bound (B) fractions were analyzed after the NaCl and EDTA chase. The graph indicates means and standard deviations from three independent experiments. (D) Specificity of second-ssDNA capture. Gel images and quantification of the protocol 1B second-DNA capture experiments in the presence of indicated ratio of nicked circular dsDNA competitor. The graph shows the means and standard deviation from three independent experiments. See also Figure S3 , showing ssDNA stimulation of the cohesin ATPase and a control that released ssDNA remains circular.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Topological but Labile ssDNA Embrace by the Cohesin Ring (A) Gel images and quantification of cohesin-loading assays using ssDNA or dsDNA as the substrate. Mis4-Ssl3 (MS) or Pds5-Wapl (PW) were added in the presence or absence of ATP. The graph shows the means and standard deviation from three independent experiments. (B) Following loading, the recovered material was challenged with NaCl and EDTA. The gel image and graph show DNA recovery at the indicated stages of the experiment. Means and standard deviations from three independent experiments are given. (C) Schematic and outcome of the dsDNA-to-ssDNA conversion experiment using E. coli exonuclease III (exoIII). Supernatant (S) and beads-bound (B) fractions were analyzed after the NaCl and EDTA chase. The graph indicates means and standard deviations from three independent experiments. (D) Specificity of second-ssDNA capture. Gel images and quantification of the protocol 1B second-DNA capture experiments in the presence of indicated ratio of nicked circular dsDNA competitor. The graph shows the means and standard deviation from three independent experiments. See also Figure S3 , showing ssDNA stimulation of the cohesin ATPase and a control that released ssDNA remains circular.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Mass Spectrometry, Standard Deviation

    Second-DNA Capture is Topological in Nature, Related to Figure 2 (A) Cohesin must topologically embrace dsDNA to mediate second-DNA capture. Protocol 1 reactions were carried out with ‘closed’ (C) or ‘linear’ (L) topology dsDNA beads. The gel image and graph show recovery of free ssDNA. The graph shows means and standard deviation from three independent experiments (WT cohesin) or the range of recovered ssDNA from two independent experiments (1B3B cohesin). (B) A DNA release experiment as shown in Figure 2 A was carried out using 1B3B cohesin. (C) Schematic of a DNA release experiment following protocol 2 s DNA capture. The ssDNA substrate was converted to dsDNA by DNA synthesis following capture. Then either of the two circular DNAs was digested with unique restriction enzymes, PstI (DNA beads) or BglII (free dsDNA). Recovered DNAs at the indicated stages of the experiment were analyzed by agarose gel electrophoresis. Input, bead bound (B) and supernatant (S) fractions are shown. (D) TEV cleavage of cohesin following second-DNA capture using protocol 2. The gel shows a representative image of input and recovered DNA, using WT and TEV cleavable (21TEV) cohesin, without or with TEV protease (TEV) treatment. After TEV protease treatment, the beads were washed with high salt buffer and recovered DNA was analyzed. The graph depicts the means and standard deviations from three independent experiments.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Second-DNA Capture is Topological in Nature, Related to Figure 2 (A) Cohesin must topologically embrace dsDNA to mediate second-DNA capture. Protocol 1 reactions were carried out with ‘closed’ (C) or ‘linear’ (L) topology dsDNA beads. The gel image and graph show recovery of free ssDNA. The graph shows means and standard deviation from three independent experiments (WT cohesin) or the range of recovered ssDNA from two independent experiments (1B3B cohesin). (B) A DNA release experiment as shown in Figure 2 A was carried out using 1B3B cohesin. (C) Schematic of a DNA release experiment following protocol 2 s DNA capture. The ssDNA substrate was converted to dsDNA by DNA synthesis following capture. Then either of the two circular DNAs was digested with unique restriction enzymes, PstI (DNA beads) or BglII (free dsDNA). Recovered DNAs at the indicated stages of the experiment were analyzed by agarose gel electrophoresis. Input, bead bound (B) and supernatant (S) fractions are shown. (D) TEV cleavage of cohesin following second-DNA capture using protocol 2. The gel shows a representative image of input and recovered DNA, using WT and TEV cleavable (21TEV) cohesin, without or with TEV protease (TEV) treatment. After TEV protease treatment, the beads were washed with high salt buffer and recovered DNA was analyzed. The graph depicts the means and standard deviations from three independent experiments.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Standard Deviation, DNA Synthesis, Agarose Gel Electrophoresis

    ssDNA, but not dsDNA, Is a Substrate for Second-Strand Capture, Related to Figure 1 (A and B) Extended gel images of Figures 1 A and 1B, showing both beads bound and supernatant fractions. 25% of the supernatant fractions are shown. (C) Protocol 2 reactions were carried out using the indicated protein concentrations (each of cohesin, Mis4-Ssl3 and Psc3). The gel image and quantification of captured dsDNA are shown. (D) A typical gel image of inputs and products of a second-DNA capture reaction following protocol 1B, performed with WT cohesin. (E) Gel images showing second-DNA capture by 1B3B cohesin, containing Walker B mutations in both Psm1 and Psm3, using a protocol 1 reaction. (F) As (E), but the reaction followed protocol 2. (G) Competition of ATP with ADP or non-hydrolyzable ATP-γ−S. As in Figure 1 E (top gel image), but an additional reaction was performed in which 0.25 mM of ATP was present in all reactions that were then supplemented by additional nucleotides. The ability of ADP and ATP-γ−S to compete with ATP demonstrates that both nucleotides are able to bind cohesin, but that their hydrolysis is required for second-DNA capture. (H) Cohesin mediates second-DNA capture irrespective of sequence homology. Protocol 2 reactions were carried out using pSKsxAS ssDNA (partially homologous to the free dsDNA substrate) or ΦX174 virion ssDNA (of unrelated sequence) as substrates. The graph presents means and standard deviations from three independent experiments.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: ssDNA, but not dsDNA, Is a Substrate for Second-Strand Capture, Related to Figure 1 (A and B) Extended gel images of Figures 1 A and 1B, showing both beads bound and supernatant fractions. 25% of the supernatant fractions are shown. (C) Protocol 2 reactions were carried out using the indicated protein concentrations (each of cohesin, Mis4-Ssl3 and Psc3). The gel image and quantification of captured dsDNA are shown. (D) A typical gel image of inputs and products of a second-DNA capture reaction following protocol 1B, performed with WT cohesin. (E) Gel images showing second-DNA capture by 1B3B cohesin, containing Walker B mutations in both Psm1 and Psm3, using a protocol 1 reaction. (F) As (E), but the reaction followed protocol 2. (G) Competition of ATP with ADP or non-hydrolyzable ATP-γ−S. As in Figure 1 E (top gel image), but an additional reaction was performed in which 0.25 mM of ATP was present in all reactions that were then supplemented by additional nucleotides. The ability of ADP and ATP-γ−S to compete with ATP demonstrates that both nucleotides are able to bind cohesin, but that their hydrolysis is required for second-DNA capture. (H) Cohesin mediates second-DNA capture irrespective of sequence homology. Protocol 2 reactions were carried out using pSKsxAS ssDNA (partially homologous to the free dsDNA substrate) or ΦX174 virion ssDNA (of unrelated sequence) as substrates. The graph presents means and standard deviations from three independent experiments.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Sequencing

    Second-DNA Capture by the Fission Yeast Cohesin Ring (A) Schematic of the second-DNA capture assay (protocol 1) and a gel image showing input and recovered DNA from the assay performed with the indicated substrates. ds, dsDNA; ss, ssDNA; rc, relaxed circular; c, circular. All reactions were carried out in the presence of ATP and an ATP regenerating system. 16.7% or 25% of input free dsDNA or ssDNA are shown. (B) Schematic of the second-DNA capture assay (protocol 2) and a representative gel image. 25% of input DNA is shown. (C and D) Quantification of the assays in (A) and (B), respectively, performed with WT and 1B3B cohesin. The means and standard deviations from three independent experiments are shown. (E) Quantification of second-DNA capture, using protocol 1B, in the absence or presence of the indicated adenosine derivatives. The means and standard deviations from three independent experiments are shown. See also Figure S1 for gel images that include supernatant fractions, reactions using 1B3B cohesin, titration of components, ATP competition, and an assay using ΦX174 ssDNA.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Second-DNA Capture by the Fission Yeast Cohesin Ring (A) Schematic of the second-DNA capture assay (protocol 1) and a gel image showing input and recovered DNA from the assay performed with the indicated substrates. ds, dsDNA; ss, ssDNA; rc, relaxed circular; c, circular. All reactions were carried out in the presence of ATP and an ATP regenerating system. 16.7% or 25% of input free dsDNA or ssDNA are shown. (B) Schematic of the second-DNA capture assay (protocol 2) and a representative gel image. 25% of input DNA is shown. (C and D) Quantification of the assays in (A) and (B), respectively, performed with WT and 1B3B cohesin. The means and standard deviations from three independent experiments are shown. (E) Quantification of second-DNA capture, using protocol 1B, in the absence or presence of the indicated adenosine derivatives. The means and standard deviations from three independent experiments are shown. See also Figure S1 for gel images that include supernatant fractions, reactions using 1B3B cohesin, titration of components, ATP competition, and an assay using ΦX174 ssDNA.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Titration