e coli rnase h Search Results


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  • 99
    New England Biolabs e coli rnaseh
    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.
    E Coli Rnaseh, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher e coli rnase h
    <t>RNase</t> H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.
    E Coli Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa e coli rnase h
    H1 RNA and protein subunits of RNase P are required for Pol I transcription in extracts. A. Whole HeLa extracts were subjected to immunodepletion analysis using 200 µL of serum containing polyclonal rabbit antibodies against Rpp20, Rpp25 or p53. Transcription reactions in the immunodepleted extracts (grey bars) or in extracts reconstituted with their corresponding immunoprecipitates (black bars) were carried out using the mini-rDNA gene and labeled RNAs were analyzed as described in Figure 1C . The 432-nt mini-rRNA band was quantitated and the optical density (in arbitrary units) was plotted. B. A proposed secondary structure of H1 RNA and the nucleotide sequence against which the antisense H1-1 deoxyoligonucleotide was directed. The upper half of H1 RNA represents the specificity domain. Conserved domains, including the P4 pseudoknot in the lower (catalytic) domain are shown. C. Whole HeLa extracts (15 mg/ml) were incubated with 8 µg of H1-1 (lane 3) or scrambled H1-1sc (lane 4) deoxyoligonucleotide in the presence of <t>RNase</t> H for 45 min as described [10] . Extracts were then assayed for RNase P activity in processing of 32 P-precursor tRNA Tyr , and cleavage products were analyzed in an 8% sequencing gel. The 5′ leader sequence (5′) and shorter species (arrow head) generated as a result of substrate miscleavage, are indicated. A concentrated DEAE-purified RNase P preparation (Ctrl; lane 2) was used as control for the correct cleavage of the substrate. D. Whole HeLa extracts described in C were subjected to transcription of the mini-rDNA gene and 5S rRNA genes as described in Figure 1C . E. Optical density of the mini-rRNA and 5S rRNA bands seen in panel D.
    E Coli Rnase H, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore e coli rnase h
    H1 RNA and protein subunits of RNase P are required for Pol I transcription in extracts. A. Whole HeLa extracts were subjected to immunodepletion analysis using 200 µL of serum containing polyclonal rabbit antibodies against Rpp20, Rpp25 or p53. Transcription reactions in the immunodepleted extracts (grey bars) or in extracts reconstituted with their corresponding immunoprecipitates (black bars) were carried out using the mini-rDNA gene and labeled RNAs were analyzed as described in Figure 1C . The 432-nt mini-rRNA band was quantitated and the optical density (in arbitrary units) was plotted. B. A proposed secondary structure of H1 RNA and the nucleotide sequence against which the antisense H1-1 deoxyoligonucleotide was directed. The upper half of H1 RNA represents the specificity domain. Conserved domains, including the P4 pseudoknot in the lower (catalytic) domain are shown. C. Whole HeLa extracts (15 mg/ml) were incubated with 8 µg of H1-1 (lane 3) or scrambled H1-1sc (lane 4) deoxyoligonucleotide in the presence of <t>RNase</t> H for 45 min as described [10] . Extracts were then assayed for RNase P activity in processing of 32 P-precursor tRNA Tyr , and cleavage products were analyzed in an 8% sequencing gel. The 5′ leader sequence (5′) and shorter species (arrow head) generated as a result of substrate miscleavage, are indicated. A concentrated DEAE-purified RNase P preparation (Ctrl; lane 2) was used as control for the correct cleavage of the substrate. D. Whole HeLa extracts described in C were subjected to transcription of the mini-rDNA gene and 5S rRNA genes as described in Figure 1C . E. Optical density of the mini-rRNA and 5S rRNA bands seen in panel D.
    E Coli Rnase H, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche e coli rnase h
    R-loop processing by human cell extract. In vitro transcription of a (CAG) 79 ·(CTG) 79 repeat-containing plasmid with [α- 32 P]rCTP was performed followed by RNase A treatment (to cleave single-stranded RNA); labeled ‘A’ or <t>RNase</t> H treatment (to also cleave RNA:DNA hybrids of the R-loop); labeled ‘H’ or human cell extract treatment; labeled ‘Ext.’ as indicated. The configuration of the R-loop generated is schematically represented above the gel. Autoradiographic signal in the gel represents R-loop formation. The position of supercoiled plasmid in dimer and monomer form is indicated by ‘sc’ where the top ‘sc’ represents linked dimers and bottom ‘sc’ represents monomers.
    E Coli Rnase H, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega e coli rnase h
    The 26-mer DNA primer annealed to the 3′ end of the 100-mer TAR RNA is extended to the base of the TAR hairpin where RT pauses. <t>RNase</t> H cleaves the RNA once. RT can copy the template to the 5′ end. RT falls off the end of the fully double-stranded RNA-DNA duplex. RT can rebind and degrade the RNA strand, leaving a 14-mer from the 5′ end of the RNA template. In the absence of NC, the 14-mer can fall off, allowing hairpin formation and self-priming. In the presence of NC, the 14-mer remains annealed, preventing hairpin formation and self-priming.
    E Coli Rnase H, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Enzymatics escherichia coli rnase h
    The 26-mer DNA primer annealed to the 3′ end of the 100-mer TAR RNA is extended to the base of the TAR hairpin where RT pauses. <t>RNase</t> H cleaves the RNA once. RT can copy the template to the 5′ end. RT falls off the end of the fully double-stranded RNA-DNA duplex. RT can rebind and degrade the RNA strand, leaving a 14-mer from the 5′ end of the RNA template. In the absence of NC, the 14-mer can fall off, allowing hairpin formation and self-priming. In the presence of NC, the 14-mer remains annealed, preventing hairpin formation and self-priming.
    Escherichia Coli Rnase H, supplied by Enzymatics, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare e coli rnase h
    E. coli <t>RNase</t> H digestion of both alleles of the 544-mer fragment of the RPA70–1,674 U/C mRNA. ( a ) Nucleotide sequence of the PS-oligos used in the assay. Oligos 1 and 2 target the polymorphic site; the bases opposing the polymorphism in the mRNA are shown in boldface. Oligos 3 – 6 target the site of different allelic structures around nucleotide 1,656. The terminal residues of oligos 1 , 2 , 3 , and 6 are labeled according to the positions of the complementary nucleotides in RPA70 mRNA. Oligos 4 and 5 share the same target hybridization sequence as 3 , but with additional nucleotides (shown underlined) added to facilitate folding into hairpin structures for intended enhancement in allele discrimination. ( b ) E. coli RNase H digestion patterns of the 5′- 32 P-labeled U and C alleles hybridized to the oligos in a . Cleavage sites were confirmed by running on the same gel RNase T1 digestion ladders for the two alleles (data not shown): they are centered at position 1,674 for oligos 1 and 2 , 1,650 for oligos 3 – 5 , and 1,654 for oligo 6 . ( c ) Comparison of percent cleavages of the U and C alleles calculated from b . Oligo-dependent cleavage bands were integrated as a percentage of total RNA in each lane with the overlapping background-cleavage bands subtracted. Variations in sample loading to each lane were internally corrected by measuring percent cleavage instead of absolute band intensity.
    E Coli Rnase H, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher ambion ribonuclease h
    E. coli <t>RNase</t> H digestion of both alleles of the 544-mer fragment of the RPA70–1,674 U/C mRNA. ( a ) Nucleotide sequence of the PS-oligos used in the assay. Oligos 1 and 2 target the polymorphic site; the bases opposing the polymorphism in the mRNA are shown in boldface. Oligos 3 – 6 target the site of different allelic structures around nucleotide 1,656. The terminal residues of oligos 1 , 2 , 3 , and 6 are labeled according to the positions of the complementary nucleotides in RPA70 mRNA. Oligos 4 and 5 share the same target hybridization sequence as 3 , but with additional nucleotides (shown underlined) added to facilitate folding into hairpin structures for intended enhancement in allele discrimination. ( b ) E. coli RNase H digestion patterns of the 5′- 32 P-labeled U and C alleles hybridized to the oligos in a . Cleavage sites were confirmed by running on the same gel RNase T1 digestion ladders for the two alleles (data not shown): they are centered at position 1,674 for oligos 1 and 2 , 1,650 for oligos 3 – 5 , and 1,654 for oligo 6 . ( c ) Comparison of percent cleavages of the U and C alleles calculated from b . Oligo-dependent cleavage bands were integrated as a percentage of total RNA in each lane with the overlapping background-cleavage bands subtracted. Variations in sample loading to each lane were internally corrected by measuring percent cleavage instead of absolute band intensity.
    Ambion Ribonuclease H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Boehringer Mannheim e coli rnase h
    Assay in vitro for oligodeoxynucleotide activity with E. coli <t>RNase</t> H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.
    E Coli Rnase H, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc e coli rnase h
    Assay in vitro for oligodeoxynucleotide activity with E. coli <t>RNase</t> H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.
    E Coli Rnase H, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Promega recombinant escherichia coli rnase h
    Assay in vitro for oligodeoxynucleotide activity with E. coli <t>RNase</t> H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.
    Recombinant Escherichia Coli Rnase H, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher escherichia coli superscript ii rnase h reverse transcriptase
    Assay in vitro for oligodeoxynucleotide activity with E. coli <t>RNase</t> H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.
    Escherichia Coli Superscript Ii Rnase H Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Roche rnase h
    Assay in vitro for oligodeoxynucleotide activity with E. coli <t>RNase</t> H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.
    Rnase H, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore e coli pyrophosphatase
    Assay in vitro for oligodeoxynucleotide activity with E. coli <t>RNase</t> H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.
    E Coli Pyrophosphatase, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Promega methods ribonuclease h
    DICER+AGO2 enzymes cleave fluorogenic substrates into duplex products that are unstable at T > 25°C. Enzymatic activity (37°C) is shown for the dsRNA DICER substrates (BoGD664AS * GD664S,  A ; BoGD664AS-dAdG * GD664S,  C ; BoPD664S * PD664AS,  E ; BoPD664S-dAdG * PD664AS,  G ) and fluorogenic siRNA BoPsi664 (BoPsi664S * Psi664AS;  I ) by AGO2 (120 nM) + DICER (30 nM) but not by  E .  coli  RNase H (0.24 U/mL). Melting analysis shows that only products of DICER+AGO2 cleavage are already melted at  T  ≥ 25°C, whereas control reactions containing either no enzyme or RNAse H instead of DICER+AGO2 have fluorimetric dsRNA stability (as measured by  T m ) that is indistinguishable from  T m  of the substrates ( B, D, F, H, J ). EDTA inhibits enzymatic cleavage of substrates and gives the characteristic decrease in  T m  compared to Assay Buffer containing divalent cations ( B, D, F, H, J ). First derivatives are displayed using dashed lines.
    Methods Ribonuclease H, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega escherichia coli rnase h1
    <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.
    Escherichia Coli Rnase H1, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Protein Sciences Inc e coli ppase
    <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 Ppase, supplied by Protein Sciences Inc, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher u e coli rnase h1
    <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.
    U E Coli Rnase H1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e coli β galactosidase
    <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.
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    Roche escherichia coli rnase hi
    <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.
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    Merck & Co e coli rnase hi
    <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.
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    TaKaRa alkaline phosphatase e coli c75
    <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.
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    New England Biolabs dna polymerase
    <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.
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    Protein Sciences Inc eschericha coli ribonuclease hi protein sci
    <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.
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    Thermo Fisher rnaseh
    <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.
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    Thermo Fisher dntp mix
    <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.
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    ATCC hela cells
    <t>NTH1-catalyzed</t> DNA glycosylase activity towards pyrimidine-derived hydantoins in <t>HeLa</t> cell extracts. 5′-[ 32 P]-labelled oligonucleotide duplexes were incubated with HeLa cell extracts under “BER+EDTA” condition. ( A ) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lane 2, as 1 but incubated with extract from HeLa cells treated with 400 nM of the non-specific siRNA; lane 3, as 1 but incubated with the extract from HeLa cells treated with 100 nM NTH1-specific siRNA; lane 4, as 3 but using 400 nM of NTH1-specific siRNA; lane 5, as 1 but with 10 nM NTH1; lane 6, as 1 but with 5 nM NEIL1; lane 7, as 1 but with 1 nM APE1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. ( B ) Western blot analysis of the siRNA-induced down-regulation of NTH1 expression in HeLa cells. Lane 1, control HeLa cells tansfected with 400 nM of the non-specific siRNA; lane 2, HeLa cells tansfected with 100 nM of NTH1-specific siRNA; lane 3, same as 2 but 400 nM siRNA; lane 4, the purified truncated recombinant ΔN-NTH1 protein. ( C ) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G and 5OH-5Me-Hyd•A in extracts. For comparison DNA repair activities on DHU•G substrate were also shown. The cleavage activities in each cell-free extract were normalized to the relative densitometry values of the actin bands on the western blot in panel B. The arrows denote the position of the 9-mer cleavage fragments containing 3′-dRP residue (9-dRP), 3′-hydroxyl group (3′-OH) and 3′-phosphate residue (3′-P), generated by NTH1, APE1 and NEIL1, respectively. For details see Materials and Methods .
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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:RNA hybrids form around DNA break sites to facilitate DNA repair in a Drosha-dependent manner. a Relocation of inactivated E. coli mCherry-RNase H1 D10R E48R to sites of laser-induced DNA damage. Representative fluorescence images, top. Scale bars, 10 µm. Bottom, graph showing quantitation of 168 cells over 3 replicates, error bars = SEM. b DNA:RNA hybrid IP (DRIP) followed by qPCR around HR and NHEJ DNA break sites, and control undamaged actin exon 5 locus after 2 h of damage induction. As a positive control, samples were treated in vitro with RNase H1 (shaded section). Error bars = SEM, Student’s paired T -test, * p ≤ 0.05 in 4 biological replicates. c DRIP-Seq was performed in conditions as in b . Graph shows enrichment of DNA:RNA hybrids around HR-repaired and NHEJ-repaired cut sites following DNA damage compared to sites documented to remain uncut following damage induction. d Over-expression of RNase H1 or a GFP control was followed by DNA resection assay as in Fig.5 a, b. N = 3, error bars = SEM, Student’s 2-sample T -test, ** p ≤ 0.01. e RNase H1 was over-expressed in the HR (left) and NHEJ (right) repair reporter system cell lines (described in Fig. 4a ) 6 h prior to I-SceI expression and GFP-positive cells were quantified as a measure of repair efficiency. N = 3 each, error bars = SD, Student’s 2-sample T -test, * p ≤ 0.05

    Journal: Nature Communications

    Article Title: Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair

    doi: 10.1038/s41467-018-02893-x

    Figure Lengend Snippet: DNA:RNA hybrids form around DNA break sites to facilitate DNA repair in a Drosha-dependent manner. a Relocation of inactivated E. coli mCherry-RNase H1 D10R E48R to sites of laser-induced DNA damage. Representative fluorescence images, top. Scale bars, 10 µm. Bottom, graph showing quantitation of 168 cells over 3 replicates, error bars = SEM. b DNA:RNA hybrid IP (DRIP) followed by qPCR around HR and NHEJ DNA break sites, and control undamaged actin exon 5 locus after 2 h of damage induction. As a positive control, samples were treated in vitro with RNase H1 (shaded section). Error bars = SEM, Student’s paired T -test, * p ≤ 0.05 in 4 biological replicates. c DRIP-Seq was performed in conditions as in b . Graph shows enrichment of DNA:RNA hybrids around HR-repaired and NHEJ-repaired cut sites following DNA damage compared to sites documented to remain uncut following damage induction. d Over-expression of RNase H1 or a GFP control was followed by DNA resection assay as in Fig.5 a, b. N = 3, error bars = SEM, Student’s 2-sample T -test, ** p ≤ 0.01. e RNase H1 was over-expressed in the HR (left) and NHEJ (right) repair reporter system cell lines (described in Fig. 4a ) 6 h prior to I-SceI expression and GFP-positive cells were quantified as a measure of repair efficiency. N = 3 each, error bars = SD, Student’s 2-sample T -test, * p ≤ 0.05

    Article Snippet: Every 500 ng genomic DNA was treated with 5 units of RNase H1 (NEB M0297) at 37 °C for 15 min. As described in Fig. , in vitro restriction digestion is required to assay for the presence of ssDNA around break sites.

    Techniques: Fluorescence, Quantitation Assay, Real-time Polymerase Chain Reaction, Non-Homologous End Joining, Positive Control, In Vitro, Over Expression, Resection Assay, Expressing

    RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Labeling

    Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Sequencing

    Truncated substrates assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent HIV-1 RT incubated with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Truncated substrates assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent HIV-1 RT incubated with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Incubation, Construct

    Truncated substrates assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent E478Q RT assayed with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Truncated substrates assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent E478Q RT assayed with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Construct

    (A) Complementation of HIV-1 RT with E. coli RNase H. Reactions were performed as described in Materials and Methods. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows. Reactions were allowed to proceed for 12 min in the presence of Mg 2+ , and then of E. coli RNase H was added (indicated by the vertical arrows). Time points are indicated above each lane in minutes. (B) Complementation of E478Q RT with E. coli RNase H. Reactions were performed as described for panel A. Time points are indicated above each lane in minutes. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: (A) Complementation of HIV-1 RT with E. coli RNase H. Reactions were performed as described in Materials and Methods. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows. Reactions were allowed to proceed for 12 min in the presence of Mg 2+ , and then of E. coli RNase H was added (indicated by the vertical arrows). Time points are indicated above each lane in minutes. (B) Complementation of E478Q RT with E. coli RNase H. Reactions were performed as described for panel A. Time points are indicated above each lane in minutes. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques:

    ) (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: ) (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques:

    H1 RNA and protein subunits of RNase P are required for Pol I transcription in extracts. A. Whole HeLa extracts were subjected to immunodepletion analysis using 200 µL of serum containing polyclonal rabbit antibodies against Rpp20, Rpp25 or p53. Transcription reactions in the immunodepleted extracts (grey bars) or in extracts reconstituted with their corresponding immunoprecipitates (black bars) were carried out using the mini-rDNA gene and labeled RNAs were analyzed as described in Figure 1C . The 432-nt mini-rRNA band was quantitated and the optical density (in arbitrary units) was plotted. B. A proposed secondary structure of H1 RNA and the nucleotide sequence against which the antisense H1-1 deoxyoligonucleotide was directed. The upper half of H1 RNA represents the specificity domain. Conserved domains, including the P4 pseudoknot in the lower (catalytic) domain are shown. C. Whole HeLa extracts (15 mg/ml) were incubated with 8 µg of H1-1 (lane 3) or scrambled H1-1sc (lane 4) deoxyoligonucleotide in the presence of RNase H for 45 min as described [10] . Extracts were then assayed for RNase P activity in processing of 32 P-precursor tRNA Tyr , and cleavage products were analyzed in an 8% sequencing gel. The 5′ leader sequence (5′) and shorter species (arrow head) generated as a result of substrate miscleavage, are indicated. A concentrated DEAE-purified RNase P preparation (Ctrl; lane 2) was used as control for the correct cleavage of the substrate. D. Whole HeLa extracts described in C were subjected to transcription of the mini-rDNA gene and 5S rRNA genes as described in Figure 1C . E. Optical density of the mini-rRNA and 5S rRNA bands seen in panel D.

    Journal: PLoS ONE

    Article Title: Function and Assembly of a Chromatin-Associated RNase P that Is Required for Efficient Transcription by RNA Polymerase I

    doi: 10.1371/journal.pone.0004072

    Figure Lengend Snippet: H1 RNA and protein subunits of RNase P are required for Pol I transcription in extracts. A. Whole HeLa extracts were subjected to immunodepletion analysis using 200 µL of serum containing polyclonal rabbit antibodies against Rpp20, Rpp25 or p53. Transcription reactions in the immunodepleted extracts (grey bars) or in extracts reconstituted with their corresponding immunoprecipitates (black bars) were carried out using the mini-rDNA gene and labeled RNAs were analyzed as described in Figure 1C . The 432-nt mini-rRNA band was quantitated and the optical density (in arbitrary units) was plotted. B. A proposed secondary structure of H1 RNA and the nucleotide sequence against which the antisense H1-1 deoxyoligonucleotide was directed. The upper half of H1 RNA represents the specificity domain. Conserved domains, including the P4 pseudoknot in the lower (catalytic) domain are shown. C. Whole HeLa extracts (15 mg/ml) were incubated with 8 µg of H1-1 (lane 3) or scrambled H1-1sc (lane 4) deoxyoligonucleotide in the presence of RNase H for 45 min as described [10] . Extracts were then assayed for RNase P activity in processing of 32 P-precursor tRNA Tyr , and cleavage products were analyzed in an 8% sequencing gel. The 5′ leader sequence (5′) and shorter species (arrow head) generated as a result of substrate miscleavage, are indicated. A concentrated DEAE-purified RNase P preparation (Ctrl; lane 2) was used as control for the correct cleavage of the substrate. D. Whole HeLa extracts described in C were subjected to transcription of the mini-rDNA gene and 5S rRNA genes as described in Figure 1C . E. Optical density of the mini-rRNA and 5S rRNA bands seen in panel D.

    Article Snippet: Whole HeLa extract (15 µl; 10–15 mg/ml; not diluted) was incubated with 8 µg of H1-1 or scrambled H1-1sc deoxyoligonucleotide (Sigma, Israel) and 40 units of E. coli RNase H (Takara Bio, Inc.) for 45 min at 30°C in a final volume of 25 µl.

    Techniques: Labeling, Sequencing, Incubation, Activity Assay, Generated, Purification

    R-loop processing by human cell extract. In vitro transcription of a (CAG) 79 ·(CTG) 79 repeat-containing plasmid with [α- 32 P]rCTP was performed followed by RNase A treatment (to cleave single-stranded RNA); labeled ‘A’ or RNase H treatment (to also cleave RNA:DNA hybrids of the R-loop); labeled ‘H’ or human cell extract treatment; labeled ‘Ext.’ as indicated. The configuration of the R-loop generated is schematically represented above the gel. Autoradiographic signal in the gel represents R-loop formation. The position of supercoiled plasmid in dimer and monomer form is indicated by ‘sc’ where the top ‘sc’ represents linked dimers and bottom ‘sc’ represents monomers.

    Journal: Nucleic Acids Research

    Article Title: Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

    doi: 10.1093/nar/gku658

    Figure Lengend Snippet: R-loop processing by human cell extract. In vitro transcription of a (CAG) 79 ·(CTG) 79 repeat-containing plasmid with [α- 32 P]rCTP was performed followed by RNase A treatment (to cleave single-stranded RNA); labeled ‘A’ or RNase H treatment (to also cleave RNA:DNA hybrids of the R-loop); labeled ‘H’ or human cell extract treatment; labeled ‘Ext.’ as indicated. The configuration of the R-loop generated is schematically represented above the gel. Autoradiographic signal in the gel represents R-loop formation. The position of supercoiled plasmid in dimer and monomer form is indicated by ‘sc’ where the top ‘sc’ represents linked dimers and bottom ‘sc’ represents monomers.

    Article Snippet: Samples were treated with either 1 μg of RNase A (Roche) alone or with 1 μg of RNase A (Roche) and 1 U of E. coli RNase H (Roche) in a final volume of 10 μl containing 1x NEB buffer #2 at room temperature for 30 min. Nucleic acid material was subsequently extracted as described above.

    Techniques: In Vitro, CTG Assay, Plasmid Preparation, Labeling, Generated

    R-loop formation in (CAG) 79 ·(CTG) 79 templates. Templates were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The position of supercoiled plasmid is indicated as ‘sc’ and open circular plasmid as ‘oc’. Products above these are catenated multimers, which also form R-loops.

    Journal: Nucleic Acids Research

    Article Title: Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

    doi: 10.1093/nar/gku658

    Figure Lengend Snippet: R-loop formation in (CAG) 79 ·(CTG) 79 templates. Templates were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The position of supercoiled plasmid is indicated as ‘sc’ and open circular plasmid as ‘oc’. Products above these are catenated multimers, which also form R-loops.

    Article Snippet: Samples were treated with either 1 μg of RNase A (Roche) alone or with 1 μg of RNase A (Roche) and 1 U of E. coli RNase H (Roche) in a final volume of 10 μl containing 1x NEB buffer #2 at room temperature for 30 min. Nucleic acid material was subsequently extracted as described above.

    Techniques: CTG Assay, In Vitro, Plasmid Preparation, Migration

    Instability analysis following R-loop removal. ( A ) Detection of S-DNA structures following R-loop removal with RNase H. DNA templates were transcribed to generate r(CAG), r(CUG) or r(CAG)+r(CUG) R-loops and then treated with RNase A and RNase H to remove all single-stranded RNA and RNA:DNA hybrids. EM was performed on individual molecules in the presence of bacterial SSB to detect unpaired DNA strands that exist in S-DNA structures (see ‘Materials and Methods’). A total of 20 molecules were analyzed for each sample type. DNA controls that were not transcribed contained 2/20 (10%) molecules bound by SSB. The number of samples bound by SSB at a single position following R-loop removal is indicated above each image and expressed as a percentage. ( B ) Percentage of unstable products following processing of RNase H-treated R-loops. R-loop products of each configuration were treated with RNase H prior to cell extract processing and assessed for instability through STRIP analysis as in (A). Products from (A) (RNase A only) were compared to RNase H-treated R-loops (RNase A+H) using the χ 2 test. Data for RNase H-treated R-loop processing are derived from three independent in vitro transcription and human cell extract processing reactions with ∼150 colonies representing 150 individual products of cell extract treatment for each RNase H-treated R-loop configuration. Specific colony numbers are as follows: rCAG-148, rCUG-152, rCAG+rCUG-153. Dashed line indicates DNA control level of instability (21%) for comparison. ( C ) Distribution of unstable products of HeLa extract processing following RNase-H-mediated R-loop removal. Sizes were determined for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described ( 26 ) and plotted. Only unstable products are shown; the stable repeat size of 79 is indicated by the dashed vertical line.

    Journal: Nucleic Acids Research

    Article Title: Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

    doi: 10.1093/nar/gku658

    Figure Lengend Snippet: Instability analysis following R-loop removal. ( A ) Detection of S-DNA structures following R-loop removal with RNase H. DNA templates were transcribed to generate r(CAG), r(CUG) or r(CAG)+r(CUG) R-loops and then treated with RNase A and RNase H to remove all single-stranded RNA and RNA:DNA hybrids. EM was performed on individual molecules in the presence of bacterial SSB to detect unpaired DNA strands that exist in S-DNA structures (see ‘Materials and Methods’). A total of 20 molecules were analyzed for each sample type. DNA controls that were not transcribed contained 2/20 (10%) molecules bound by SSB. The number of samples bound by SSB at a single position following R-loop removal is indicated above each image and expressed as a percentage. ( B ) Percentage of unstable products following processing of RNase H-treated R-loops. R-loop products of each configuration were treated with RNase H prior to cell extract processing and assessed for instability through STRIP analysis as in (A). Products from (A) (RNase A only) were compared to RNase H-treated R-loops (RNase A+H) using the χ 2 test. Data for RNase H-treated R-loop processing are derived from three independent in vitro transcription and human cell extract processing reactions with ∼150 colonies representing 150 individual products of cell extract treatment for each RNase H-treated R-loop configuration. Specific colony numbers are as follows: rCAG-148, rCUG-152, rCAG+rCUG-153. Dashed line indicates DNA control level of instability (21%) for comparison. ( C ) Distribution of unstable products of HeLa extract processing following RNase-H-mediated R-loop removal. Sizes were determined for each unstable product of processing from electrophoretic migration position relative to known size markers as previously described ( 26 ) and plotted. Only unstable products are shown; the stable repeat size of 79 is indicated by the dashed vertical line.

    Article Snippet: Samples were treated with either 1 μg of RNase A (Roche) alone or with 1 μg of RNase A (Roche) and 1 U of E. coli RNase H (Roche) in a final volume of 10 μl containing 1x NEB buffer #2 at room temperature for 30 min. Nucleic acid material was subsequently extracted as described above.

    Techniques: Stripping Membranes, Derivative Assay, In Vitro, Migration

    C9orf72 repeat forms R-loops and double-R-loops. ( A ) Templates with (GGGGCC) 60 ·(GGCCCC) 60 were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The slower migrating products above these are catenated multimers, which also form R-loops. ( B ) Templates with 13, 21 or 60 C9orf72 repeats were transcribed as in panel (A) to reveal single and double-R-loop formation.

    Journal: Nucleic Acids Research

    Article Title: Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability

    doi: 10.1093/nar/gku658

    Figure Lengend Snippet: C9orf72 repeat forms R-loops and double-R-loops. ( A ) Templates with (GGGGCC) 60 ·(GGCCCC) 60 were in vitro transcribed with T3 and/or T7 RNA polymerases and treated with RNase A (which digests single-stranded RNA) to form each R-loop configuration indicated schematically above the gel. The presence of R-loops forces the plasmid into a more open configuration, thus reducing electrophoretic migration within the gel. Treatment of the R-loop with RNase H cleaves RNA that is base-paired to DNA (the RNA:DNA hybrid) and thus collapses the R-loop, returning DNA to supercoiled form. The slower migrating products above these are catenated multimers, which also form R-loops. ( B ) Templates with 13, 21 or 60 C9orf72 repeats were transcribed as in panel (A) to reveal single and double-R-loop formation.

    Article Snippet: Samples were treated with either 1 μg of RNase A (Roche) alone or with 1 μg of RNase A (Roche) and 1 U of E. coli RNase H (Roche) in a final volume of 10 μl containing 1x NEB buffer #2 at room temperature for 30 min. Nucleic acid material was subsequently extracted as described above.

    Techniques: In Vitro, Plasmid Preparation, Migration

    The 26-mer DNA primer annealed to the 3′ end of the 100-mer TAR RNA is extended to the base of the TAR hairpin where RT pauses. RNase H cleaves the RNA once. RT can copy the template to the 5′ end. RT falls off the end of the fully double-stranded RNA-DNA duplex. RT can rebind and degrade the RNA strand, leaving a 14-mer from the 5′ end of the RNA template. In the absence of NC, the 14-mer can fall off, allowing hairpin formation and self-priming. In the presence of NC, the 14-mer remains annealed, preventing hairpin formation and self-priming.

    Journal: Journal of Virology

    Article Title: In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing

    doi: 10.1128/JVI.75.2.672-686.2001

    Figure Lengend Snippet: The 26-mer DNA primer annealed to the 3′ end of the 100-mer TAR RNA is extended to the base of the TAR hairpin where RT pauses. RNase H cleaves the RNA once. RT can copy the template to the 5′ end. RT falls off the end of the fully double-stranded RNA-DNA duplex. RT can rebind and degrade the RNA strand, leaving a 14-mer from the 5′ end of the RNA template. In the absence of NC, the 14-mer can fall off, allowing hairpin formation and self-priming. In the presence of NC, the 14-mer remains annealed, preventing hairpin formation and self-priming.

    Article Snippet: A large amount of R-75 accumulated by 15 s, as was described above, but in the presence of heparin, most of the R-75 did not undergo further processing to yield the 65-base single-stranded RNA unless E. coli RNase H was introduced into the reaction (after 5 min).

    Techniques:

    The 5′ 14-base RNA remains annealed to −sssDNA only in the presence of NC. 26 DNA primer was annealed to 5′-end-labeled TAR-100 RNA and extended by RT in the presence (lanes 2 to 13) or absence (lanes 15 to 26) of NC. At the indicated times, aliquots of the reactions were added to a mixture of heparin (to sequester RT) and E. coli RNase H (to digest RNA annealed to DNA). After incubation with E. coli RNase H, the reactions were stopped by the addition of formamide gel loading buffer, and the samples were loaded onto a 12% denaturing polyacrylamide gel and fractionated by electrophoresis. The sizes of DNA and RNA are shown on the right of the figure. Lanes 1 and 14 are control lanes (marked C) showing untreated TAR-100 RNA.

    Journal: Journal of Virology

    Article Title: In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing

    doi: 10.1128/JVI.75.2.672-686.2001

    Figure Lengend Snippet: The 5′ 14-base RNA remains annealed to −sssDNA only in the presence of NC. 26 DNA primer was annealed to 5′-end-labeled TAR-100 RNA and extended by RT in the presence (lanes 2 to 13) or absence (lanes 15 to 26) of NC. At the indicated times, aliquots of the reactions were added to a mixture of heparin (to sequester RT) and E. coli RNase H (to digest RNA annealed to DNA). After incubation with E. coli RNase H, the reactions were stopped by the addition of formamide gel loading buffer, and the samples were loaded onto a 12% denaturing polyacrylamide gel and fractionated by electrophoresis. The sizes of DNA and RNA are shown on the right of the figure. Lanes 1 and 14 are control lanes (marked C) showing untreated TAR-100 RNA.

    Article Snippet: A large amount of R-75 accumulated by 15 s, as was described above, but in the presence of heparin, most of the R-75 did not undergo further processing to yield the 65-base single-stranded RNA unless E. coli RNase H was introduced into the reaction (after 5 min).

    Techniques: Labeling, Incubation, Electrophoresis

    E. coli RNase H digestion of both alleles of the 544-mer fragment of the RPA70–1,674 U/C mRNA. ( a ) Nucleotide sequence of the PS-oligos used in the assay. Oligos 1 and 2 target the polymorphic site; the bases opposing the polymorphism in the mRNA are shown in boldface. Oligos 3 – 6 target the site of different allelic structures around nucleotide 1,656. The terminal residues of oligos 1 , 2 , 3 , and 6 are labeled according to the positions of the complementary nucleotides in RPA70 mRNA. Oligos 4 and 5 share the same target hybridization sequence as 3 , but with additional nucleotides (shown underlined) added to facilitate folding into hairpin structures for intended enhancement in allele discrimination. ( b ) E. coli RNase H digestion patterns of the 5′- 32 P-labeled U and C alleles hybridized to the oligos in a . Cleavage sites were confirmed by running on the same gel RNase T1 digestion ladders for the two alleles (data not shown): they are centered at position 1,674 for oligos 1 and 2 , 1,650 for oligos 3 – 5 , and 1,654 for oligo 6 . ( c ) Comparison of percent cleavages of the U and C alleles calculated from b . Oligo-dependent cleavage bands were integrated as a percentage of total RNA in each lane with the overlapping background-cleavage bands subtracted. Variations in sample loading to each lane were internally corrected by measuring percent cleavage instead of absolute band intensity.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Single-nucleotide polymorphisms can cause different structural folds of mRNA

    doi:

    Figure Lengend Snippet: E. coli RNase H digestion of both alleles of the 544-mer fragment of the RPA70–1,674 U/C mRNA. ( a ) Nucleotide sequence of the PS-oligos used in the assay. Oligos 1 and 2 target the polymorphic site; the bases opposing the polymorphism in the mRNA are shown in boldface. Oligos 3 – 6 target the site of different allelic structures around nucleotide 1,656. The terminal residues of oligos 1 , 2 , 3 , and 6 are labeled according to the positions of the complementary nucleotides in RPA70 mRNA. Oligos 4 and 5 share the same target hybridization sequence as 3 , but with additional nucleotides (shown underlined) added to facilitate folding into hairpin structures for intended enhancement in allele discrimination. ( b ) E. coli RNase H digestion patterns of the 5′- 32 P-labeled U and C alleles hybridized to the oligos in a . Cleavage sites were confirmed by running on the same gel RNase T1 digestion ladders for the two alleles (data not shown): they are centered at position 1,674 for oligos 1 and 2 , 1,650 for oligos 3 – 5 , and 1,654 for oligo 6 . ( c ) Comparison of percent cleavages of the U and C alleles calculated from b . Oligo-dependent cleavage bands were integrated as a percentage of total RNA in each lane with the overlapping background-cleavage bands subtracted. Variations in sample loading to each lane were internally corrected by measuring percent cleavage instead of absolute band intensity.

    Article Snippet: E. coli RNase H (0.6 units, Amersham Pharmacia) was then added to the renatured mRNA and incubated for 5 min at 37°C.

    Techniques: Sequencing, Labeling, Hybridization

    Assay in vitro for oligodeoxynucleotide activity with E. coli RNase H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.

    Journal: BMC Molecular Biology

    Article Title: Expression of c-myc is not critical for cell proliferation in established human leukemia lines

    doi: 10.1186/1471-2199-2-13

    Figure Lengend Snippet: Assay in vitro for oligodeoxynucleotide activity with E. coli RNase H against c- myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc -specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c- myc mRNA.

    Article Snippet: In vitro RNase H assays Total RNA isolated from KYO1 cells was diluted to a concentration of 1 μg/18 μl reaction volume in complete First Strand Buffer mix (GibcoBRL) containing 0.4 nmol oligodeoxynucleotide and 0.5 U E. coli RNase H (Boehringer Mannheim UK Ltd, Lewes, East Sussex, UK).

    Techniques: In Vitro, Activity Assay, Isolation, Incubation, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis

    Effects of an RNase H-inactive morpholino antisense oligonucleotide on c- myc  mRNA, c-Myc protein, and proliferation of KYO1 cells. The antisense, and control sense and nonsense oligonucleotides (Table   1 ) were delivered into cells by streptolysin O permeabilization from an external concentration of 20 μM. (A) Upper panels; Northern blots of mRNA at 4 and 24 h. Lower panels; Western blots of c-Myc protein at 4 and 24 h. The 47 kDa truncated protein containing the c-Myc epitope recognised by the antibody mix resulted from initiation of translation at an in-frame AUG 300 bases downstream of the c-Myc 2 AUG initiation codon [  22 ]. (B) Cell number relative to that at 2 h after loading cells with oligonucleotide. Values are the mean and standard deviation of 3 replicates.

    Journal: BMC Molecular Biology

    Article Title: Expression of c-myc is not critical for cell proliferation in established human leukemia lines

    doi: 10.1186/1471-2199-2-13

    Figure Lengend Snippet: Effects of an RNase H-inactive morpholino antisense oligonucleotide on c- myc mRNA, c-Myc protein, and proliferation of KYO1 cells. The antisense, and control sense and nonsense oligonucleotides (Table 1 ) were delivered into cells by streptolysin O permeabilization from an external concentration of 20 μM. (A) Upper panels; Northern blots of mRNA at 4 and 24 h. Lower panels; Western blots of c-Myc protein at 4 and 24 h. The 47 kDa truncated protein containing the c-Myc epitope recognised by the antibody mix resulted from initiation of translation at an in-frame AUG 300 bases downstream of the c-Myc 2 AUG initiation codon [ 22 ]. (B) Cell number relative to that at 2 h after loading cells with oligonucleotide. Values are the mean and standard deviation of 3 replicates.

    Article Snippet: In vitro RNase H assays Total RNA isolated from KYO1 cells was diluted to a concentration of 1 μg/18 μl reaction volume in complete First Strand Buffer mix (GibcoBRL) containing 0.4 nmol oligodeoxynucleotide and 0.5 U E. coli RNase H (Boehringer Mannheim UK Ltd, Lewes, East Sussex, UK).

    Techniques: Concentration Assay, Northern Blot, Western Blot, Standard Deviation

    Comparison of the chimeric c- myc  antisense 20-mer, 5'F-MD757AS (Table   1 ) with the CpG 5-mer motif CGTTG contained within its phosphodiester section for effects on c- myc  mRNA, c-Myc protein and proliferation of KYO1 cells. Cells were loaded with oligodeoxynucleotide by streptolysin O permeabilization from an external concentration of 20 μM. (A) Cell proliferation over 24 h. (B) Levels of full length c- myc  mRNA 30 min after oligodeoxynucleotide delivery. RNase H-generated mRNA fragments were visible on the Northern blot for cells treated with the 20-mer but not the 5-mer. (C) Relative levels of c-Myc protein. Values in all panels are the mean and standard deviation of 3 replicates.

    Journal: BMC Molecular Biology

    Article Title: Expression of c-myc is not critical for cell proliferation in established human leukemia lines

    doi: 10.1186/1471-2199-2-13

    Figure Lengend Snippet: Comparison of the chimeric c- myc antisense 20-mer, 5'F-MD757AS (Table 1 ) with the CpG 5-mer motif CGTTG contained within its phosphodiester section for effects on c- myc mRNA, c-Myc protein and proliferation of KYO1 cells. Cells were loaded with oligodeoxynucleotide by streptolysin O permeabilization from an external concentration of 20 μM. (A) Cell proliferation over 24 h. (B) Levels of full length c- myc mRNA 30 min after oligodeoxynucleotide delivery. RNase H-generated mRNA fragments were visible on the Northern blot for cells treated with the 20-mer but not the 5-mer. (C) Relative levels of c-Myc protein. Values in all panels are the mean and standard deviation of 3 replicates.

    Article Snippet: In vitro RNase H assays Total RNA isolated from KYO1 cells was diluted to a concentration of 1 μg/18 μl reaction volume in complete First Strand Buffer mix (GibcoBRL) containing 0.4 nmol oligodeoxynucleotide and 0.5 U E. coli RNase H (Boehringer Mannheim UK Ltd, Lewes, East Sussex, UK).

    Techniques: Concentration Assay, Generated, Northern Blot, Standard Deviation

    The effects of an RNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotide on c- myc  mRNA and c-Myc protein in KYO1 cells. (A) Relative levels of mRNA and protein at 4 h post intracytoplasmic delivery by streptolysin O permeabilization were determined by densitometry of Northern and Western blots. Data represent the mean and standard deviation of 3 replicates. The structures of the antisense oligodeoxynucleotide, and inverted antisense and sense controls are given in Table   1 . (B) Northern blots of c- myc  mRNA at 4 h following oligodeoxynucleotide delivery by streptolysin O permeabilization and by electroporation of KYO1 cells. Grey arrow; full length mRNA. Black arrows; RNase H-generated 5' fragments of mRNA.

    Journal: BMC Molecular Biology

    Article Title: Expression of c-myc is not critical for cell proliferation in established human leukemia lines

    doi: 10.1186/1471-2199-2-13

    Figure Lengend Snippet: The effects of an RNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotide on c- myc mRNA and c-Myc protein in KYO1 cells. (A) Relative levels of mRNA and protein at 4 h post intracytoplasmic delivery by streptolysin O permeabilization were determined by densitometry of Northern and Western blots. Data represent the mean and standard deviation of 3 replicates. The structures of the antisense oligodeoxynucleotide, and inverted antisense and sense controls are given in Table 1 . (B) Northern blots of c- myc mRNA at 4 h following oligodeoxynucleotide delivery by streptolysin O permeabilization and by electroporation of KYO1 cells. Grey arrow; full length mRNA. Black arrows; RNase H-generated 5' fragments of mRNA.

    Article Snippet: In vitro RNase H assays Total RNA isolated from KYO1 cells was diluted to a concentration of 1 μg/18 μl reaction volume in complete First Strand Buffer mix (GibcoBRL) containing 0.4 nmol oligodeoxynucleotide and 0.5 U E. coli RNase H (Boehringer Mannheim UK Ltd, Lewes, East Sussex, UK).

    Techniques: Northern Blot, Western Blot, Standard Deviation, Electroporation, Generated

    DICER+AGO2 enzymes cleave fluorogenic substrates into duplex products that are unstable at T > 25°C. Enzymatic activity (37°C) is shown for the dsRNA DICER substrates (BoGD664AS * GD664S,  A ; BoGD664AS-dAdG * GD664S,  C ; BoPD664S * PD664AS,  E ; BoPD664S-dAdG * PD664AS,  G ) and fluorogenic siRNA BoPsi664 (BoPsi664S * Psi664AS;  I ) by AGO2 (120 nM) + DICER (30 nM) but not by  E .  coli  RNase H (0.24 U/mL). Melting analysis shows that only products of DICER+AGO2 cleavage are already melted at  T  ≥ 25°C, whereas control reactions containing either no enzyme or RNAse H instead of DICER+AGO2 have fluorimetric dsRNA stability (as measured by  T m ) that is indistinguishable from  T m  of the substrates ( B, D, F, H, J ). EDTA inhibits enzymatic cleavage of substrates and gives the characteristic decrease in  T m  compared to Assay Buffer containing divalent cations ( B, D, F, H, J ). First derivatives are displayed using dashed lines.

    Journal: PLoS ONE

    Article Title: DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

    doi: 10.1371/journal.pone.0120614

    Figure Lengend Snippet: DICER+AGO2 enzymes cleave fluorogenic substrates into duplex products that are unstable at T > 25°C. Enzymatic activity (37°C) is shown for the dsRNA DICER substrates (BoGD664AS * GD664S, A ; BoGD664AS-dAdG * GD664S, C ; BoPD664S * PD664AS, E ; BoPD664S-dAdG * PD664AS, G ) and fluorogenic siRNA BoPsi664 (BoPsi664S * Psi664AS; I ) by AGO2 (120 nM) + DICER (30 nM) but not by E . coli RNase H (0.24 U/mL). Melting analysis shows that only products of DICER+AGO2 cleavage are already melted at T ≥ 25°C, whereas control reactions containing either no enzyme or RNAse H instead of DICER+AGO2 have fluorimetric dsRNA stability (as measured by T m ) that is indistinguishable from T m of the substrates ( B, D, F, H, J ). EDTA inhibits enzymatic cleavage of substrates and gives the characteristic decrease in T m compared to Assay Buffer containing divalent cations ( B, D, F, H, J ). First derivatives are displayed using dashed lines.

    Article Snippet: Materials and Methods Ribonuclease H (Escherichia coli ) was from Promega (Madison, WI).

    Techniques: Activity Assay

    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

    Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.

    Journal: PLoS Pathogens

    Article Title: Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys

    doi: 10.1371/journal.ppat.1008262

    Figure Lengend Snippet: Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.

    Article Snippet: PCR, RACE and qPCR from MKPV RNA For splice site confirmations, MKPV-infected kidney RNA was treated or mock-treated with Turbo DNase (Thermo Fisher, 0.4 U/μg RNA) and ExoI (New England Biolabs, 2 U/μg RNA) for 30 min at 37˚C, followed by incubation with DNase Inactivation Reagent (Thermo Fisher, 0.2ul/ μg RNA).

    Techniques: Sequencing, Produced, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Purification

    NTH1-catalyzed DNA glycosylase activity towards pyrimidine-derived hydantoins in HeLa cell extracts. 5′-[ 32 P]-labelled oligonucleotide duplexes were incubated with HeLa cell extracts under “BER+EDTA” condition. ( A ) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lane 2, as 1 but incubated with extract from HeLa cells treated with 400 nM of the non-specific siRNA; lane 3, as 1 but incubated with the extract from HeLa cells treated with 100 nM NTH1-specific siRNA; lane 4, as 3 but using 400 nM of NTH1-specific siRNA; lane 5, as 1 but with 10 nM NTH1; lane 6, as 1 but with 5 nM NEIL1; lane 7, as 1 but with 1 nM APE1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. ( B ) Western blot analysis of the siRNA-induced down-regulation of NTH1 expression in HeLa cells. Lane 1, control HeLa cells tansfected with 400 nM of the non-specific siRNA; lane 2, HeLa cells tansfected with 100 nM of NTH1-specific siRNA; lane 3, same as 2 but 400 nM siRNA; lane 4, the purified truncated recombinant ΔN-NTH1 protein. ( C ) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G and 5OH-5Me-Hyd•A in extracts. For comparison DNA repair activities on DHU•G substrate were also shown. The cleavage activities in each cell-free extract were normalized to the relative densitometry values of the actin bands on the western blot in panel B. The arrows denote the position of the 9-mer cleavage fragments containing 3′-dRP residue (9-dRP), 3′-hydroxyl group (3′-OH) and 3′-phosphate residue (3′-P), generated by NTH1, APE1 and NEIL1, respectively. For details see Materials and Methods .

    Journal: PLoS ONE

    Article Title: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways

    doi: 10.1371/journal.pone.0021039

    Figure Lengend Snippet: NTH1-catalyzed DNA glycosylase activity towards pyrimidine-derived hydantoins in HeLa cell extracts. 5′-[ 32 P]-labelled oligonucleotide duplexes were incubated with HeLa cell extracts under “BER+EDTA” condition. ( A ) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lane 2, as 1 but incubated with extract from HeLa cells treated with 400 nM of the non-specific siRNA; lane 3, as 1 but incubated with the extract from HeLa cells treated with 100 nM NTH1-specific siRNA; lane 4, as 3 but using 400 nM of NTH1-specific siRNA; lane 5, as 1 but with 10 nM NTH1; lane 6, as 1 but with 5 nM NEIL1; lane 7, as 1 but with 1 nM APE1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. ( B ) Western blot analysis of the siRNA-induced down-regulation of NTH1 expression in HeLa cells. Lane 1, control HeLa cells tansfected with 400 nM of the non-specific siRNA; lane 2, HeLa cells tansfected with 100 nM of NTH1-specific siRNA; lane 3, same as 2 but 400 nM siRNA; lane 4, the purified truncated recombinant ΔN-NTH1 protein. ( C ) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G and 5OH-5Me-Hyd•A in extracts. For comparison DNA repair activities on DHU•G substrate were also shown. The cleavage activities in each cell-free extract were normalized to the relative densitometry values of the actin bands on the western blot in panel B. The arrows denote the position of the 9-mer cleavage fragments containing 3′-dRP residue (9-dRP), 3′-hydroxyl group (3′-OH) and 3′-phosphate residue (3′-P), generated by NTH1, APE1 and NEIL1, respectively. For details see Materials and Methods .

    Article Snippet: Cell culture and silencing of APE1 and NTH1 expression HeLa cells (ATCC #CCL-2, U.S.A.) were routinely grown at 37°C in 5% CO2 in Dulbecco minimal essential medium supplemented with 10% foetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin.

    Techniques: Activity Assay, Derivative Assay, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Purification, Recombinant, Generated