xrn1  (New England Biolabs)


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    Structured Review

    New England Biolabs xrn1
    Both knocking-down and knocking-out of <t>XRN1</t> had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

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    New England Biolabs xrn1
    Both knocking-down and knocking-out of <t>XRN1</t> had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
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    New England Biolabs arh tbb 2 3
    Microinjection of liposome-DNA complexes facilitates expression of the P eef-1A.1 ::TurboRFP:: tbb-2 -3’UTR transgene in  A. rhodensis . Expression was primarily limited to late embryogenesis in F1 progeny. Scale bar 50 µM.
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    New England Biolabs t4 dna ligase
    Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with <t>T4</t> DNA ligase before transfection into Huh7 cells. The uncut dimer
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    Image Search Results


    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Journal: PLoS ONE

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    doi: 10.1371/journal.pone.0201250

    Figure Lengend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Article Snippet: The DENV sfRNA is readily formed when treated with XRN1 at concentration as low as 0.01 units ( , lane 7), whereas the 800-nt JEV RNA was relatively resistant to low concentrations of XRN1 (0.01 and 0.1 unit) ( , lanes 2 and 3).

    Techniques: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Journal: PLoS ONE

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    doi: 10.1371/journal.pone.0201250

    Figure Lengend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Article Snippet: The DENV sfRNA is readily formed when treated with XRN1 at concentration as low as 0.01 units ( , lane 7), whereas the 800-nt JEV RNA was relatively resistant to low concentrations of XRN1 (0.01 and 0.1 unit) ( , lanes 2 and 3).

    Techniques: Synthesized

    Microinjection of liposome-DNA complexes facilitates expression of the P eef-1A.1 ::TurboRFP:: tbb-2 -3’UTR transgene in  A. rhodensis . Expression was primarily limited to late embryogenesis in F1 progeny. Scale bar 50 µM.

    Journal: bioRxiv

    Article Title: Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems

    doi: 10.1101/429126

    Figure Lengend Snippet: Microinjection of liposome-DNA complexes facilitates expression of the P eef-1A.1 ::TurboRFP:: tbb-2 -3’UTR transgene in A. rhodensis . Expression was primarily limited to late embryogenesis in F1 progeny. Scale bar 50 µM.

    Article Snippet: The Cel-rpl-23 3’ UTR was removed by digestion with SacI and EcoRI and replaced with the Arh-tbb-2 3’UTR by Gibson cloning, according to the manufacturer’s instructions (NEB).

    Techniques: Expressing

    Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer

    Journal: Journal of Clinical Microbiology

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    doi: 10.1128/JCM.02340-10

    Figure Lengend Snippet: Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer

    Article Snippet: In fact, the uncut plasmid DNA expressed much higher levels of L, M, and S proteins than the BspQI digest whether or not the HBV genome was further circularized by T4 DNA ligase ( , compare lanes 1, 2, and 3).

    Techniques: Transfection

    Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and

    Journal: Journal of Clinical Microbiology

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    doi: 10.1128/JCM.02340-10

    Figure Lengend Snippet: Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and

    Article Snippet: In fact, the uncut plasmid DNA expressed much higher levels of L, M, and S proteins than the BspQI digest whether or not the HBV genome was further circularized by T4 DNA ligase ( , compare lanes 1, 2, and 3).

    Techniques: Functional Assay, Polymerase Chain Reaction, Clone Assay, Transfection

    Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Article Snippet: Consistent with the inability of HMO2 to protect DNA with BspHI-generated 5′ overhangs, such DNA may be re-ligated in presence of T4 DNA ligase ( B, lane 3; lanes 1 and 2 contain DNA without and with T4 DNA ligase, respectively).

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Article Snippet: Consistent with the inability of HMO2 to protect DNA with BspHI-generated 5′ overhangs, such DNA may be re-ligated in presence of T4 DNA ligase ( B, lane 3; lanes 1 and 2 contain DNA without and with T4 DNA ligase, respectively).

    Techniques: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Article Snippet: Consistent with the inability of HMO2 to protect DNA with BspHI-generated 5′ overhangs, such DNA may be re-ligated in presence of T4 DNA ligase ( B, lane 3; lanes 1 and 2 contain DNA without and with T4 DNA ligase, respectively).

    Techniques:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Article Snippet: Consistent with the inability of HMO2 to protect DNA with BspHI-generated 5′ overhangs, such DNA may be re-ligated in presence of T4 DNA ligase ( B, lane 3; lanes 1 and 2 contain DNA without and with T4 DNA ligase, respectively).

    Techniques: Sequencing, Incubation, Ligation