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    New England Biolabs rnase h
    RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate  rnh1 / 201∆  cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note  rad50∆  and  rnh1 / 201∆  cells form smaller colonies, indicating increased cell death. In transient exposure assays,  rnh1 / 201∆  cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate  rnh1 / 201∆  cells are moderately sensitive to HU. In transient exposure assays,  rnh1 / 201∆  cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.
    Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2919 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs dna polymerase
    RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate  rnh1 / 201∆  cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note  rad50∆  and  rnh1 / 201∆  cells form smaller colonies, indicating increased cell death. In transient exposure assays,  rnh1 / 201∆  cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate  rnh1 / 201∆  cells are moderately sensitive to HU. In transient exposure assays,  rnh1 / 201∆  cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 3856 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/New England Biolabs
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    95
    New England Biolabs e coli exoi
    <t>DNA</t> with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli <t>ExoI.</t> The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    E Coli Exoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate  rnh1 / 201∆  cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note  rad50∆  and  rnh1 / 201∆  cells form smaller colonies, indicating increased cell death. In transient exposure assays,  rnh1 / 201∆  cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate  rnh1 / 201∆  cells are moderately sensitive to HU. In transient exposure assays,  rnh1 / 201∆  cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: RNase H‐deficient cells are weakly sensitive to camptothecin Plating assays indicate rnh1 / 201∆ cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note rad50∆ and rnh1 / 201∆ cells form smaller colonies, indicating increased cell death. In transient exposure assays, rnh1 / 201∆ cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments. Plating assays indicate rnh1 / 201∆ cells are moderately sensitive to HU. In transient exposure assays, rnh1 / 201∆ cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Cycling Probe Technology, Incubation, Standard Deviation

    RNA–DNA hybrids are not enriched at the reparable mat1 DSB Diagram of the mat2,3∆ broken replication fork showing the location of PCR products used for ChIP and DRIP assays. Rad52 is enriched at the mat1 DSB site and the sister chromatid region used for HDR. ChIP assay was performed with Rad52‐5FLAG expressed from the endogenous locus in a mat2,3∆ strain. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments. RNA–DNA hybrids are enriched at the tRNATyr gene, but not at the reparable DSB at the mat1 locus in rnh1 / 201∆ cells. DRIP assay was performed with S9.6 antibody using the indicated strains with or without RNase H treatment. Relative enrichment was calculated as ChIP/input. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: RNA–DNA hybrids are not enriched at the reparable mat1 DSB Diagram of the mat2,3∆ broken replication fork showing the location of PCR products used for ChIP and DRIP assays. Rad52 is enriched at the mat1 DSB site and the sister chromatid region used for HDR. ChIP assay was performed with Rad52‐5FLAG expressed from the endogenous locus in a mat2,3∆ strain. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments. RNA–DNA hybrids are enriched at the tRNATyr gene, but not at the reparable DSB at the mat1 locus in rnh1 / 201∆ cells. DRIP assay was performed with S9.6 antibody using the indicated strains with or without RNase H treatment. Relative enrichment was calculated as ChIP/input. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Polymerase Chain Reaction, Chromatin Immunoprecipitation

    HDR‐mediated reset of collapsed replication forks is essential in RNase H‐deficient cells Tetrad analysis showing that Rad50 is essential in  rnh1 / 201∆  background. Tetrad analysis showing that Ctp1 is essential in  rnh1 / 201∆  background. Tetrad analysis showing that Mus81 is essential in  rnh1 / 201∆  background.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: HDR‐mediated reset of collapsed replication forks is essential in RNase H‐deficient cells Tetrad analysis showing that Rad50 is essential in rnh1 / 201∆ background. Tetrad analysis showing that Ctp1 is essential in rnh1 / 201∆ background. Tetrad analysis showing that Mus81 is essential in rnh1 / 201∆ background.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques:

    RNase H‐deficient cells are insensitive to ionizing radiation RNase H is not required for IR survival. Cells exposed to IR from a cesium‐137 source were plated with fivefold serial dilutions. Plates were photographed after 3‐day incubation at 32°C. Quantitative analysis confirms that rnh1 / 201∆ cells are insensitive to IR. Bars represent standard deviation of three independent biological experiments.

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: RNase H‐deficient cells are insensitive to ionizing radiation RNase H is not required for IR survival. Cells exposed to IR from a cesium‐137 source were plated with fivefold serial dilutions. Plates were photographed after 3‐day incubation at 32°C. Quantitative analysis confirms that rnh1 / 201∆ cells are insensitive to IR. Bars represent standard deviation of three independent biological experiments.

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Incubation, Standard Deviation

    DSB repair at the mat1 broken replication fork occurs efficiently in the absence of RNase H Mating type switching system in fission yeast. See text for details. Lower panel shows repair mechanism in mat2,3∆ donorless strain. Proficient mating type switching in h 90 rnh1 / 201∆ cells. Colonies of the indicated genotypes on SSA plates were exposed to iodine vapor to assess mating/sporulation efficiency. Controls include wild‐type h 90 , non‐switchable h − , and switching‐defective swi3∆ h 90 . Rad50 is required SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rad50∆ mat2,3∆ cells display very poor growth compared to single mutants or wild type. Products of a tetrad dissection were photographed on successive days. RNase H is not required for SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rnh1 / 201∆ mat2,3∆ cells display no growth defect relative to rnh1∆ rnh201∆ .

    Journal: EMBO Reports

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

    doi: 10.15252/embr.201745335

    Figure Lengend Snippet: DSB repair at the mat1 broken replication fork occurs efficiently in the absence of RNase H Mating type switching system in fission yeast. See text for details. Lower panel shows repair mechanism in mat2,3∆ donorless strain. Proficient mating type switching in h 90 rnh1 / 201∆ cells. Colonies of the indicated genotypes on SSA plates were exposed to iodine vapor to assess mating/sporulation efficiency. Controls include wild‐type h 90 , non‐switchable h − , and switching‐defective swi3∆ h 90 . Rad50 is required SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rad50∆ mat2,3∆ cells display very poor growth compared to single mutants or wild type. Products of a tetrad dissection were photographed on successive days. RNase H is not required for SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rnh1 / 201∆ mat2,3∆ cells display no growth defect relative to rnh1∆ rnh201∆ .

    Article Snippet: Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Techniques: Dissection

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw274

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

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

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw274

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

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

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